啤酒花(Humulus lupulus L.)是釀造啤酒的基本原料之一，其中iso-humulones (iso-α-acids)和humulinones是主要苦味的來源。近年來許多酒廠會在啤酒發酵階段加入啤酒花以達到更具啤酒花特性的風味，也使得humulones (α-acids)產生更多不同的苦味化合物衍生物。在本實驗中，針對60種的啤酒花苦味化合物開發液相層析串聯質譜分析方法，以鹼性的甲酸銨溶液與甲醇/乙腈(70/30)作為層析條件能在25分鐘內將分析物分離。此方法線性範圍在0.053-3912 ng/mL之間，並且相關係數皆大於0.9938，iso-α-acids和humulinones的LOQ分別為0.26 ng/mL和0.053 ng/mL。本實驗也有良好的精密度(RSD< 0.5%)和準確度(recovery 86.3%-118.1%)。針對兩款啤酒Vienna Lager、Double-India Pale Ale在發酵的過程與儲存的過程進行監測，其定量結果顯示未經由酒花乾投(Dry-hopping)的Vienna Lager較傾向於產生質子催化、環化的的苦味衍生物，而有進行酒花乾投的DIPA較傾向由氧化產生的苦味衍生物。此結果也顯示釀造原料、釀造手法等對於啤酒花苦味化合物產生的傾向有不同的影響。

Hops (Humulus lupulus L.) are essential raw materials for beer brewing, and the major contributors to beer bitterness are iso-humulones (iso-α-acids) and humulinones. In recent years, many breweries have focused on the production of hop-forward beer style by adding hops after or during the cold fermentation stage, which will tend to release humulinones or other hop-derived bitter compounds. In this study, a LC-MS/MS method was developed for quantification of 60 hop-derived bitter compounds in 25 minutes. A reverse phase chromatography with alkaline methanol/acetonitrile (70/30) mobile phase was used for the separation. The quantitative range were 0.053-3912 ng/mL with correlation coefficient r> 0.99, and the LOQ were 0.26 and 0.053 ng/mL for iso-α-acids and humulinones. Precision (RSD< 5.0%) and accuracy (recovery 86.3%-118.1%) were both satisfactory. The abundance of hop-derived bitter compounds in the dry-hopping beer Double-India Pale Ale and the non-dry-hopping beer (Vienna Lager) were monitored throughout the fermentation and storage stages, the formation of oxidation and cyclization products showed difference profiles between these two beers. The quantification results reveal how hop-derived bitter compounds change throughout the brewing process, as well as the influence of hops and brewing techniques on beer bitterness.

[1] Bamforth CW. Brewing materials and processes: A practical approach to beer excellence: Academic Press; 2016.
[2] Takoi K. Flavor Hops” varieties and various flavor compounds contributing to their “varietal aromas”: A review. MBAA Tech Q 2019;56:113-23.
[3] Lafontaine S, Caffrey A, Dailey J, Varnum S, Hale A, Eichler B, Dennenlöhr J, Schubert C, Knoke L, Lerno L. Evaluation of variety, maturity, and farm on the concentrations of monoterpene diglycosides and hop volatile/nonvolatile composition in five humulus lupulus cultivars. Journal of Agricultural and Food Chemistry 2021;69:4356-70.
[4] Sanekata A, Tanigawa A, Takoi K, Nakayama Y, Tsuchiya Y. Identification and characterization of geranic acid as a unique flavor compound of hops (Humulus lupulus L.) variety Sorachi Ace. Journal of Agricultural and Food Chemistry 2018;66:12285-95.
[5] Dietz C, Cook D, Huismann M, Wilson C, Ford R. The multisensory perception of hop essential oil: A review. Journal of the Institute of Brewing 2020;126:320-42.
[6] Malowicki MG, Shellhammer TH. Isomerization and degradation kinetics of hop (Humulus lupulus) acids in a model wort-boiling system. Journal of Agricultural and Food Chemistry 2005;53:4434-9.
[7] Stevens R. The chemistry of hop constituents. Chemical Reviews 1967;67:19-71.
[8] Dresel M, Vogt C, Dunkel A, Hofmann T. The bitter chemodiversity of hops (Humulus lupulus L.). Journal of Agricultural and Food Chemistry 2016;64:7789-99.
[9] Taniguchi Y, Taniguchi H, Yamada M, Matsukura Y, Koizumi H, Furihata K, Shindo K. Analysis of the components of hard resin in hops (Humulus lupulus L.) and structural elucidation of their transformation products formed during the brewing process. Journal of Agricultural and Food Chemistry 2014;62:11602-12.
[10] Taniguchi Y, Yamada M, Taniguchi H, Matsukura Y, Shindo K. Chemical characterization of beer aging products derived from hard resin components in hops (Humulus lupulus L.). Journal of Agricultural and Food Chemistry 2015;63:10181-91.
[11] Biendl M, Cocuzza S. Hard Resins–new findings about a familiar hop fraction. Hopfenrundsch Int 2016;2017:59-68.
[12] Van Opstaele F, De Rouck G, Janssens P, Montandon G. An exploratory study on the impact of the yeast strain on hop flavour expressions in heavily hopped beers: New England IPA. Brew Sci 2020;73:26-40.
[13] Lafontaine SR, Shellhammer TH. How hoppy beer production has redefined hop quality and a discussion of agricultural and processing strategies to promote it. MBAA Tech Q 2019;56:1-12.
[14] Maye JP, Smith R. Hidden secrets of the New England IPA. MBAA Tech Q 2018;55:88-92.
[15] Biendl M, Schmidt C, Maye JP, Smith R. New England IPA: The Hop Aroma Champion of Beers. MBAA Tech Q 2021.
[16] Takoi K, Itoga Y, Takayanagi J, Matsumoto I, Nakayama Y. Control of hop aroma impression of beer with blend-hopping using geraniol-rich hop and new hypothesis of synergy among hop-derived flavour compounds. Brew Sci 2016;69:85-93.
[17] Kishimoto T, Wanikawa A, Kono K, Shibata K. Comparison of the odor-active compounds in unhopped beer and beers hopped with different hop varieties. Journal of Agricultural and Food Chemistry 2006;54:8855-61.
[18] Hahn CD, Lafontaine SR, Pereira CB, Shellhammer TH. Evaluation of nonvolatile chemistry affecting sensory bitterness intensity of highly hopped beers. Journal of Agricultural and Food Chemistry 2018;66:3505-13.
[19] Takoi K, Tokita K, Sanekata A, Usami Y, Itoga Y, Koie K, Nakayama Y. Varietal difference of hop-derived flavour compounds in late-hopped/dry-hopped beers. Monatsschrift für Brauwissenschaft 2016;69.
[20] Kemp O, Hofmann S, Braumann I, Jensen S, Fenton A, Oladokun O. Changes in key hop‐derived compounds and their impact on perceived dry‐hop flavour in beers after storage at cold and ambient temperature. Journal of the Institute of Brewing 2021;127:367-84.
[21] Lafontaine SR, Shellhammer TH. Investigating the factors impacting aroma, flavor, and stability in dry-hopped beers. MBAA Tech Q 2019;56:13-23.
[22] Lafontaine SR, Shellhammer TH. Impact of static dry‐hopping rate on the sensory and analytical profiles of beer. Journal of the Institute of Brewing 2018;124:434-42.
[23] Hauser DG, Simaeys KRV, Lafontaine SR, Shellhammer TH. A comparison of single-stage and two-stage dry-hopping regimes. Journal of the American Society of Brewing Chemists 2019;77:251-60.
[24] Janish S. Dry hop best practices: Using science as a guide for process and recipe development. MBAA Tech Q 2021;58:59-64.
[25] Cottrell MT. A Search for Diastatic Enzymes Endogenous to Humulus lupulus and Produced by Microbes Associated with Pellet Hops Driving “Hop Creep” of Dry Hopped Beer. Journal of the American Society of Brewing Chemists 2022:1-13.
[26] Takoi K, Itoga Y, Koie K, Kosugi T, Shimase M, Katayama Y, Nakayama Y, Watari J. The contribution of geraniol metabolism to the citrus flavour of beer: synergy of geraniol and β‐citronellol under coexistence with excess linalool. Journal of the Institute of Brewing 2010;116:251-60.
[27] BJCP. 2021 Beer Style Guidelines. 2021.
[28] Krogerus K, Gibson BR. 125th anniversary review: diacetyl and its control during brewery fermentation. Journal of the Institute of Brewing 2013;119:86-97.
[29] Metrulas LK, McNeil C, Slupsky CM, Bamforth CW. The application of metabolomics to ascertain the significance of prolonged maturation in the production of lager‐style beers. Journal of the Institute of Brewing 2019;125:242-9.
[30] Leker J, Maye JP. Discovery of Acetohumulone and Acetolupulone a New Hop Alpha Acid and Beta Acid. Journal of the American Society of Brewing Chemists 2023;81:276-81.
[31] Algazzali V, Shellhammer T. Bitterness intensity of oxidized hop acids: Humulinones and hulupones. Journal of the American Society of Brewing Chemists 2016;74:36-43.
[32] Maye JP, Smith R, Leker J. Humulinone formation in hops and hop pellets and its implications for dry hopped beers. MBAA Tech Q 2016;53:23-7.
[33] Taniguchi Y, Taniguchi H, Matsukura Y, Kawachi Y, Shindo K. Structural elucidation of humulone autoxidation products and analysis of their occurrence in stored hops. Journal of Natural Products 2014;77:1252-61.
[34] Huang Y, Tippmann J, Becker T. Kinetic modeling of hop acids during wort boiling. International Journal of Bioscience, Biochemistry and Bioinformatics 2013;3:47.
[35] Intelmann D, Haseleu G, Hofmann T. LC-MS/MS quantitation of hop-derived bitter compounds in beer using the ECHO technique. Journal of Agricultural and Food Chemistry 2009;57:1172-82.
[36] Intelmann D, Demmer O, Desmer N, Hofmann T. 18O stable isotope labeling, quantitative model experiments, and molecular dynamics simulation studies on the trans-specific degradation of the bitter tasting iso-α-acids of beer. Journal of Agricultural and Food Chemistry 2009;57:11014-23.
[37] Intelmann D, Kummerlöwe G, Haseleu G, Desmer N, Schulze K, Fröhlich R, Frank O, Luy B, Hofmann T. Structures of storage‐induced transformation products of the beer’s bitter principles, revealed by sophisticated NMR spectroscopic and LC–MS techniques. Chemistry–A European Journal 2009;15:13047-58.
[38] Intelmann D, Hofmann T. On the autoxidation of bitter-tasting iso-α-acids in beer. Journal of Agricultural and Food Chemistry 2010;58:5059-67.
[39] García-Villalba R, Cortacero-Ramírez S, Segura-Carretero A, Martín-Lagos Contreras JA, Fernández-Gutiérrez A. Analysis of hop acids and their oxidized derivatives and iso-α-acids in beer by capillary electrophoresis− electrospray ionization mass spectrometry. Journal of Agricultural and Food Chemistry 2006;54:5400-9.
[40] Taniguchi Y, Matsukura Y, Ozaki H, Nishimura K, Shindo K. Identification and quantification of the oxidation products derived from α-acids and β-acids during storage of hops (Humulus lupulus L.). Journal of Agricultural and Food Chemistry 2013;61:3121-30.
[41] Haseleu G, Intelmann D, Hofmann T. Identification and RP-HPLC-ESI-MS/MS quantitation of bitter-tasting β-acid transformation products in beer. Journal of Agricultural and Food Chemistry 2009;57:7480-9.
[42] Haseleu G, Intelmann D, Hofmann T. Structure determination and sensory evaluation of novel bitter compounds formed from β-acids of hop (Humulus lupulus L.) upon wort boiling. Food Chemistry 2009;116:71-81.
[43] Dušek M, Olšovská J, Krofta K, Jurková M, Mikyška A. Qualitative determination of β-acids and their transformation products in beer and hop using HR/AM-LC-MS/MS. Journal of Agricultural and Food Chemistry 2014;62:7690-7.
[44] Howard G. Institute of brewing analysis committee estimation of the bitterness of beer. Journal of the Institute of Brewing 1968;74:249-51.
[45] ASBC. Beer Method 23A: Bitterness units (International Method). ASBC Methods of Analysis 2018.
[46] Maye JP, Smith R. Dry hopping and its effects on the international bitterness unit test and beer bitterness. MBAA Tech Q 2016;53:134-6.
[47] Cocuzza S, Zarnkow M, Stallforth A, Peifer F, Jacob F. The impact of dry hopping on selected physical and chemical attributes of beer. Brew Sci 2019;72:118-24.
[48] Forster A, Gahr A, Schüll F. What are Auxiliary Bitter Compounds in Hops and how do they Affect the Quality of Bitterness in Beer. Brew Sci 2017;70:203-9.
[49] Silva Ferreira C, Thibault de Chanvalon E, Bodart E, Collin S. Why humulinones are key bitter constituents only after dry hopping: comparison with other Belgian styles. Journal of the American Society of Brewing Chemists 2018;76:236-46.
[50] Verzele M. 100 years of hop chemistry and its relevance to brewing. Journal of the Institute of Brewing 1986;92:32-48.
[51] Hofta P, Dostálek P, Sýkora D. Liquid chromatography‐diode array and electrospray high‐accuracy mass spectrometry of iso‐α‐acids in DCHA‐Iso standard and beer. Journal of the Institute of Brewing 2007;113:48-54.
[52] Ferreira CS, Collin S. Fate of bitter compounds through dry-hopped beer aging. Why cis-humulinones should be as feared as trans-isohumulones? Journal of the American Society of Brewing Chemists 2020;78:103-13.
[53] Hao J, Speers R, Fan H, Deng Y, Dai Z. A review of cyclic and oxidative bitter derivatives of alpha, iso-alpha and beta-hop acids. Journal of the American Society of Brewing Chemists 2020;78:89-102.
[54] Huang YC. Quantification of bitterness compounds in beers using LC-MS. National Taiwan Normal University, Taipei 2020.
[55] Almeida AMd, Castel-Branco MM, Falcão A. Linear regression for calibration lines revisited: weighting schemes for bioanalytical methods. Journal of Chromatography B 2002;774:215-22.
[56] Van Holle A, Muylle H, Haesaert G, Naudts D, De Keukeleire D, Roldán‐Ruiz I, Van Landschoot A. Relevance of hop terroir for beer flavour. Journal of the Institute of Brewing 2021;127:238-47.
[57] Haseleu G, Lagemann A, Stephan A, Intelmann D, Dunkel A, Hofmann T. Quantitative sensomics profiling of hop-derived bitter compounds throughout a full-scale beer manufacturing process. Journal of Agricultural and Food Chemistry 2010;58:7930-9.
[58] Maye JP, Smith R, Leker J. Dry Hopping and Its Effect on Beer Bitterness, the IBU Test, and pH. BrauW Int 2018;2018:25-9.
[59] Justus A. Tracking IBU through the brewing process: the quest for consistency. MBAA Tech Q 2018;55:67-74.
[60] Wang L, Hong K, Agbaka JI, Zhu G, Lv C, Ma C. Application of UHPLC-Q/TOF-MS-based metabolomics analysis for the evaluation of bitter-tasting Krausen metabolites during beer fermentation. Journal of Food Composition and Analysis 2021;99:103850.
[61] De Cooman L, Aerts G, Overmeire H, De Keukeleire D. Alterations of the profiles of iso‐α‐acids during beer ageing, marked instability of trans‐iso‐α‐acids and implications for beer bitterness consistency in relation to tetrahydroiso‐α‐acids. Journal of the Institute of Brewing 2000;106:169-78.
[62] Intelmann D, Haseleu G, Dunkel A, Lagemann A, Stephan A, Hofmann T. Comprehensive sensomics analysis of hop-derived bitter compounds during storage of beer. Journal of Agricultural and Food Chemistry 2011;59:1939-53.