Analysis of Hop Acids and Their Oxidized Derivatives and Iso-α-acids in Beer by Capillary Electrophoresis−Electrospray Ionization Mass Spectrometry

Analysis of Hop Acids and Their Oxidized Derivatives and Iso-α-acids in 1 Beer by Capillary Electrophoresis−Electrospray Ionization Mass 2 Spectrometry 3 4 Rocío García-Villalba,1 Sonia Cortacero-Ramírez,2 Antonio Segura-Carretero,1* José Antonio 5 Martín-Lagos Contreras,2 Alberto Fernández-Gutiérrez1 6 7 1Department of Analytical Chemistry, Faculty of Sciences, University of Granada, c/ 8 Fuentenueva s/n, 18071 Granada, Spain, and Research Laboratories of 9 2Grupo Cervezas Alhambra S.L., Avenida Murcia 1, 18010 Granada, Spain 10 11 *Author to whom correspondence should be addressed: Dr. A. Segura Carretero or A. 12 Fernández Gutiérrez, Research Group FQM-297, Department of Analytical Chemistry, 13 Faculty of Sciences, University of Granada, C/Fuentenueva s/n, E-18071 Granada, Spain. 14 E-mail: [email protected] or [email protected]. Fax: +34 958 249510 15 16 Abstract 17 This study investigates the applicability of on-line coupling of capillary electrophoresis with 18 electrospray ionization tandem mass spectrometry (CZE-ESI-MS) for the separation and 19 characterization of α and β-acids and oxidized hop acids from crude extracts of different hop 20 varieties. CZE-ESI-MS with negative-ion electrospray ionization proved to be a suitable 21 technique for the determination of these types of natural compounds and their oxidized 22 derivatives. The CZE parameters (pH, concentration, and buffer type) and ESI-MS parameters 23 (nature and flow rate of the sheath liquid, nebulizer pressure, drying gas flow rate, 24 temperature, and compound stability) were optimized. The optimized method provides the 25 potential for a fast qualitative determination of hop acids and their oxidation compounds. The 26 method was also applied to the determination of iso-α-acids in beer. 27 28 KEYWORDS: Acetone hop extract; hop acids; iso-α-acids; capillary zone electrophoresis; 29 mass spectrometry 30 31 INTRODUCTION 32 Extracts of hop cones, the female flowers of Humulus lupulus L., are used for adding aroma 33 and flavor in the beer-brewing process. Hops contain hundreds of components but of 34 particular interest are the so-called resins, containing mainly hop acids, hop oil, and 35 polyphenols. These three classes of resin are important as biochemical markers to differentiate 36 hop varieties. The hop acids, part of the soft resin fraction, consist of two related series, the 37 α-acids (humulone, cohumulone, and adhumulone) and the β-acids (lupulone, colupulone, and 38 adlupulone) (1). Besides the two series of normal-, co-, and ad-homologues there are also 39 some minor hop acids in the plant, including posthumulone/postlupulone, 40 prehumulone/prelupulone and adprehumulone (2). These are present as a complex mixture of 41 varying composition and concentrations. The relative proportions of α-acids and β-acids as 42 well as the content of co-homologues depend on the hop variety and, for any given variety, on 43 the growing conditions. Many different varieties of the H. lupulus L. species exist, each one 44 with its own different composition and agronomic characteristics. Traditionally, hop varieties 45 have been classified into two groups, namely, “aroma” or “bitter” types, depending on their 46 α-acid content and flavor characteristics. 47 Hops are prone to oxidation and chemical deterioration. Both the α-and β-acids are very 48 susceptible to oxidation and degradation during storage. Their oxidation products affect beer 49 flavor significantly because once α-acids have been oxidized, they can no longer be 50 isomerized into iso-α-acids; thus, the hops’ bittering potential decreases, and the aroma 51 becomes unpleasant and “cheesy”. It is also important to package the hops properly, which 52 involves keeping them in refrigerated storage at temperatures of between 0 and 5 °C, 53 removing as much oxygen as possible, and storing them in an oxygen barrier material (3, 4). 54 Apart from the storage conditions, each variety has a particular tendency to be oxidized, and 55 so the oxidation state of hops is an important quality factor that needs to be looked at closely 56 for quality control in the brewing industry. 57 During the brewing process the virtually insoluble α-acids of the hop extract are converted 58 into the more soluble iso-α-acids, which give the typical bitter taste to the beer. In addition to 59 imparting bitter taste, iso-α-acids exhibit other interesting features: they have tensioactive 60 properties, thereby stabilizing the beer foam, and they inhibit the growth of Gram-positive 61 bacteria. An analysis of the hop acids in hops is important for quality control, and many 62 methods have been developed to provide a quantitative analysis of the α-acids in hops and 63 hop products. A widely used empirical method extracts the bitter components into solvents 64 and measures them quantitatively by spectrophotometry. HPLC with UV detection is also 65 routinely used to analyze bitter acids (5-12). Nevertheless, UV is neither sensitive nor 66 selective enough for the direct identification of minor hop acids in complex mixtures. The 67 instability and structural similarity of the bitter hop acids cause difficulty in routine analysis. 68 Recently, the detection of these compounds by HPLC coupled to mass spectrometry was 69 investigated. The six major bitter hop acids have been analyzed by HPLC coupled with 70 atmospheric pressure ionization tandem mass spectrometry (APCI-MS-MS) (2) and with 71 negative electrospray ionization mass spectrometry (13). Other techniques such as capillary 72 electrophoresis (CE) in its different modes have been applied to the analysis of hop acids, that 73 is, capillary zone electrophoresis (CZE) (14), micellar electrokinetic chromatography 74 (MEKC) (15, 16), and microemulsion electrokinetic chromatography (MEEKC) (17-19), 75 using a UV detector. The iso-α-acids have also been determined using MEKC-UV (20-23). 76 The aim of this work has been to develop the first fast and simple capillary 77 electrophoresis-electrospray ionization mass spectrometry (CE-ESI-MS) method for the 78 identification of hop acids and their oxidation compounds in four varieties of hops: Saaz, 79 Nugget, Magnum, and Columbus. We have also determined iso-α-acids in beer to 80 demonstrate the applicability of this method. 81 82 MATERIALS AND METHODS 83 Chemicals. The hop acid standard, an international calibration extract ICE 2, composed of a 84 mixture of α-acids (34.94% humulone + adhumulone and 14.45% cohumulone) and β-acids 85 (12.02% lupulone + adlupulone and 12.92% colupulone), and the iso-α-acid standard ICS-I2, 86 with a mixture of 64.3% trans-iso-R-acids (iso-humulone, isoadhumulone, and 87 iso-cohumulone) were from Labor Veritas, Zürich, Switzerland. 88 Ammonium acetate, ammonium carbonate, acetic acid, and diethylamine were from Panreac 89 (Barcelona, Spain), ethanolamine and diethanolamine were from Aldrich (Steinheim, 90 Germany), and ammonia was from Merck (Darmstadt, Germany), all of which were used for 91 the CE running buffers at different concentrations and pH values. Buffers were prepared by 92 weighing the quantity indicated in doubly distilled water and adding 2 M ammonium 93 hydroxide to adjust the pH. Triethylamine (TEA) was from Aldrich, and HPLC grade 94 2-propanol used in the sheath flow, acetone, and sodium hydroxide were from Panreac. All 95 solutions were filtered through 0.45 µm Millipore (Bedford, MA) membrane filters before 96 being injected into the capillary. Distilled water was deionized using a Milli-Q system 97 (Millipore). DSC-Diol and DSC-C18 solid-phase separation (SPE) cartridges were from 98 Supelco (Bellefonte, PA). 99 100 Instrumentation. For CE separations we used a P/ACE System MDQ (Beckman 101 Instruments, Fullerton, CA) equipped with a UV-visible detector and a 0-30 kV high-voltage 102 built-in power supply. A bare fused-silica capillary with 50 µm i.d. was from Composite 103 Metal Services (Worcester, U.K.). The detection length to the UV detector was 7 cm, and the 104 total length was 100 cm (corresponding to the MS detection length). The instrument was 105 controlled by a PC running System 32 Karat software from Beckman. 106 CE was coupled using an electrospray interface (ESI) (model G1607A from Agilent 107 Technologies, Palo Alto, CA) to the MS detector (Bruker Daltonics, Squire 2000). A 108 commercial coaxial sheath-flow interface was used (vide infra). The coaxial sheath liquid and 109 the electrical contact at the electrospray needle tip were delivered by a 74900-00-05 Cole 110 Palmer syringe pump (Vernon Hills, IL). An ESI-MS interface provided both a coaxial sheath 111 liquid makeup flow and a nebulization gas to assist droplet formation. Both the drying gas and 112 the nebulization gas were nitrogen. The mass spectrometer was used in the negative-ion 113 mode, and the capillary voltage was set at 4000 V. The ion trap scanned within the m/z 114 300-700 range at 13000 u/s during separation and detection in the scan mode. The maximum 115 accumulation time for the ion trap was set at 5.00 ms, the target count was set at 20000, and 116 the trap drive level was set at 100%. For the connection between the CE system and the 117 electrospray ion source of the mass spectrometer the outlet of the separation capillary was 118 fitted into the electrospray needle of the ion source, and a flow of conductive sheath liquid 119 made electrical contact between the capillary effluent and water for the electrospray needle. 120 The instrument was controlled by a PC running Esquire NT software from Bruker Daltonics. 121 Before the first use, the uncoated capillaries were conditioned using a rinse with 0.1 M NaOH 122 for 10 min followed by a rinse with water for 5 min and finally a rinse with running buffer for 123 30 min. Capillary conditioning between runs was carried out by flushing the column for 3 min 124 with water and finally for 5 min with the separation buffer. At the end of the day the capillary 125 was rinsed with water for 30 min and dried for 10 min. 126 127 Hop Samples and Beer. Hop pellets of the varieties Saaz and Nugget and bottles of “extra” 128 beer were obtained from the company Grupo Cervezas Alhambra S.L. (Granada, Spain), 129 whereas the varieties Columbus and Magnum were provided by S.A. Española de Fomento 130 del Lúpulo (León, Spain). 131 It is known that drying temperatures >65 °C cause variable losses of hop acids (3), so we 132 induced the oxidation of the α-and β-acid standards and four hop varieties: Saaz, Nugget, 133 Columbus, and Magnum. The hop pellets were received in intact, lightproof packaging. Ten 134 grams was reduced to powder with a mortar and heated for 2h at 80 °C in an oven. We also 135 studied the natural oxidation of Saaz and Nugget by keeping pellets of the harvest of the year 136 2000 in a plastic vessel at room temperature and in the presence of light for 2 years. 137 138 Extraction of Hop Acids and Oxidized Derivatives of the Hop Pellets. To recover the hop 139 acids present in the pellets and their oxidized derivatives, we studied different organic 140 solvents (methanol, ethanol, and acetone) with water (0:100, 25:75, 50:50, 75:25, and 100:0). 141 The extraction protocol was as follows: 2.5 g of hop pellets, previously reduced to powder 142 with a mortar, was extracted three times with 50 mL of acetone/water (75:25 v/v) for 10 min 143 each time. The extracts were combined and brought to dryness in a rotary evaporator under 144 reduced pressure at 60 °C to get rid of any residual solvent. During the last step, 2 mL of 145 acetone/water (50:50 v/v) was added to dissolve the extract, which was then passed through a 146 0.20 µm membrane filter and analyzed by CE-ESI-MS. 147 The extraction protocol of the naturally oxidized derivatives and the compounds obtained by 148 induced oxidation was the same for both methods and for all four varieties of hops studied. 149 150 Extraction of Isomerized Hop Acids in “Extra” Beer. We assayed two different cartridges, 151 DSC-Diol and DSC-C18, and obtained the best results with DSC-C18. Thus, the subsequent 152 extraction protocol of isomerized hop acids was as follows: a DSC-C18 cartridge placed in a 153 vacuum elution apparatus was conditioned by passing 20 mL of acetone/water (75:25 v/v) and 154 then 10 mL of water through it. Subsequently, 100 mL of degassed “extra” beer was passed 155 through the column. The isomerized hop acids were recovered by passing four portions of 5 156 mL of acetone/water (75:25 v/v). The final volume was dried in a rotary evaporator under 157 reduced pressure at 60 °C. The residue was reconstituted in 2 mL of acetone/water (50:50 v/v) 158 and passed through a 0.20 µm filter before CE-ESI-MS analysis. 159 160 General Procedure. The optimum conditions used for the CE-ESIMS separation method 161 were as follows: running buffer, 160 mM ammonium carbonate/ammonium hydroxide; pH 9; 162 voltage, 20 kV; 7 s injection time; sheath liquid, 2-propanol/water, 50:50 v/v, with 0.1% TEA 163 delivered at a flow rate of 3 µL/min; drying gas flow rate, 4 L/min at 150 °C; nebulizing gas 164 pressure, 6 psi; and MS analyses carried out using a compound stability of 25%. 165 RESULTS AND DISCUSSION 166 Development of the CE-ESI-MS Method. The effects of different separation parameters 167 were studied to obtain the best selectivity, sensitivity, and resolution conditions. The 168 CE-ESI-MS method was optimized using the extract obtained with 75:25 v/v acetone/water 169 from oxidized Saaz hops because this extract was the most complex and its electropherogram 170 presented the greatest number of peaks. First, the optimum concentration and pH of four 171 volatile running buffers, ammonium carbonate/ammonium hydroxide, diethylamine/am172 monium hydroxide, ammonium hydroxide/acetic acid, and ammonium acetate/ammonium 173 hydroxide, were established. The pH of ammonium carbonate/ammonium hydroxide was 174 assayed between 8.5 and 10 at a concentration of 100 mM, and pH 9 showed the best 175 resolution. The concentration was then studied between 100 and 190 mM at pH 9, the best 176 resolution being found with 160 mM. 177 After studying the influence of pH between 9.5 and 10.5 at a concentration of 100 mM, we 178 chose pH 10.5 as optimum. Diethylamine/ammonium hydroxide was assayed in the range 179 between 100 and 500 mM, and the best resolution was found to occur at a concentration of 180 500 mM. The pH and concentration using ammonium hydroxide/acetic acid as running buffer 181 were also examined. The effect of pH was studied by using a concentration of 500 mM of 182 ammonium hydroxide and adjusting the pH with acetic acid to between 9 and 10. We chose 183 pH 9.5 as the optimum value, and the concentration was then assayed between 100 and 500 184 mM at this pH. Higher concentrations were tried, but these produced noises in the baseline. 185 The pH of ammonium acetate/ammonium hydroxide was assayed by varying it between 8 and 186 11 when using a concentration of 160 mM. The greatest number of peaks appeared at pH 187 10.5. We then studied the concentration effect between 80 and 180 mM and found the best 188 result with a concentration of 160 mM. Figure 1 shows the optimum electropherograms 189 found in the different studies carried out with the four running buffers under optimum 190 conditions. Of all of the conditions studied, ammonium carbonate/ammonium hydroxide at 191 pH 9 at a concentration of 160 mM offered the most information about the compounds of 192 interest. 193 To obtain better resolution between peaks, different percentages (5, 10, and 15%) of organic 194 solvents such as 2-propanol and sodium dodecyl sulfate (SDS) at 5 and 10 mM were added to 195 the buffer without success. The voltage was varied between 10 and 30 kV, and finally a 196 voltage of 20 kV was selected to obtain the best resolution. The injections were made at the 197 anodic end using N2 pressure of 0.5 psi for 7 s (1psi=6894.76 Pa). These conditions were 198 chosen for the optimization of the ESI parameters. 199 It is well-known that the choice of sheath liquid has significant effects on sensitivity and on 200 the electrical contact between CE and ESI (24, 25). Generally, a small amount of volatile 201 TEA is used for ESI-negative detection (26). Next, the sheath liquid composition and flow 202 rate were optimized to increase the MS sensitivity of the compounds. Different sheath liquids 203 were tested, that is, methanol/water and 2-propanol/water at different proportions, with 0.1% 204 TEA and without TEA. With 2-propanol as organic solvent we obtained a better response than 205 with methanol. Eight different percentages of sheath flow liquid were tested: 206 2-propanol/water at 40:60 v/v, 50:50 v/v, 60:40 v/v, and 70:30 v/v, with and without 0.1% v/v 207 TEA in order to facilitate electrical contact when the negative mode was used. A 50:50 ratio 208 of propanol/water with 0.1% v/v TEA as sheath liquid was judged to obtain the highest signal 209 and best current stability. The choice of these variables represented a compromise between 210 maintaining efficient electrophoretic separation and improving ionization performance. The 211 influence of the sheath liquid flow rates of 1, 2, 3, 4, and 5 µL/min was also examined. We 212 observed that the best results in terms of MS sensitivity were obtained when using a flow rate 213 of 3 µL/min. The nebulizer pressure was then optimized by testing values of 2, 4, 6, 8, and 10 214 psi, the greatest sensitivity being obtained with 6 psi. The temperature of the interface was 215 also optimized between 100 and 300 °C, the greatest sensitivity being obtained at 150 °C. 216 Another important parameter of the interface was the stability of the compound, which was 217 studied between 25 and 100%. The MS signal decreased concomitantly with higher 218 percentages because the number of molecules transferred into MS was low, whereas with 219 lower percentages the majority of the compounds became more stable, as indicated by an 220 increase in the MS signal. This parameter is related to the voltage used in the capillary placed 221 at the MS entrance; thus, the higher this parameter, the higher the voltage applied by the MS 222 instrument and therefore the higher the solute fragmentation that can take place at that point. 223 Thus, we chose 25% compound stability. 224 Figure 2 shows the base peak electropherogram and the extracted ion electropherogram 225 obtained under the CE-ESI-MS conditions chosen for an extract of oxidized Saaz hops. 226 Fifteen different compounds can be recognized: m/z 377.3 corresponds to 227 humulinone/adhumulinone (overlap), oxidation products of humulone/adhumulone. Oxidation 228 occurs leading to the creation of a double acyloin entity. The acyloin rearrangement with 229 concurrent ring contraction may take place at both C-4 and C-6. This reaction is totally 230 analogous to the important isomerization reaction of humulone to the isohumulones (Figure 231 3a); an ion with a ratio m/z of 393.3 indicates that two oxygen atoms have been incorporated 232 into humulone/adhumulone (overlap). This oxidation product belongs to the 233 abeo-isohumulone group, which is derived from isohumulones but obtained directly through 234 the oxidation of humulone. It is very likely that humulinone represents the first step in the 235 reaction sequence. These oxidized compounds have a five-membered structure. The reaction 236 mechanism of these compounds (Figure 3b) proceeds via the oxidation of the 237 3-methyl-2-butenyl side chains in humulinone, followed by cyclization, via either 238 intramolecular dehydration or nucleophilic cleavage of the intermediate oxirane ring (1). The 239 ions with m/z ratios of 409.2 and 425.2 are thought to be more highly oxidized compounds, 240 thus indicating that three and four oxygen atoms, respectively, have been incorporated into 241 humulone/adhumulone, but their structure and the formation mechanism are still unknown; 242 m/z 363.3 corresponds to cohumulinone, an oxidation product of cohumulone, with a structure 243 and oxidation mechanism similar to that of humulinone, and the m/z of 379.2 could be due to 244 the incorporation of two oxygen atoms into cohumulone, in the same way as with humulone; 245 m/z 331.2, hulupone/adhulupone (overlap), and m/z 317.2, cohulupone, correspond to the 246 oxidation of the t-acids lupulone/ adlupulone and colupulone. The structures of the oxidation 247 products indicate that the lengths of the side chains in these compounds, together with the 248 double bonds and hydroxyl groups, easily give rise to oxidation cyclizations, leading to five 249 derivatives (Figure 3c) (m/z 375.2, prehumulone). Other compounds have been found in this 250 extract: m/z 341.1, maltose; m/z 577.2, procyanidin; m/z 609.2, hesperedin (18.6 min) and 251 rutin (19.5 min); m/z 447.1, luteolin-7-O-glucoside (17.2 min) and kaempferol-3-O-glucoside 252 (20.5 min); m/z 463.1, quercetin4′-O-glucoside; and m/z 337.1, desmethylxanthohumol. Some 253 of them have easily been identified and confirmed using the hop acid standard. 254 The reproducibility of the CE-ESI-MS analysis, expressed by the relative standard deviation 255 (RSD) of six consecutive injections, was 3.6% for the retention time and 6.9% for the peak 256 area, both quite suitable for the intentions of this work. 257 258 Analysis of Acids in Different Hop Varieties. To demonstrate the capacity of the 259 CE-ESI-MS method for the analysis of this type of compound in hop samples, we applied the 260 method to different varieties. Four varieties were studied: Saaz (a classic variety with good 261 aroma but poor storage stability); Nugget and Magnum (with similar properties of high 262 α-acid, acceptable aroma, and good storage stability); and Columbus (with high α-acid, a 263 strong but pleasant aroma, but poor storage stability). 264 CAPTION FIGURES Figure 1. Comparison between different running buffers: (a) ammonium carbonate/ammonium hydroxide, 160 mM at pH 9; (b) diethylamine/ammonium hydroxide, 500 mM at pH 10.5; (c) ammonium hydroxide/acetic acid, 500 mM at pH 9.5; (d) ammonium acetate/ammonium hydroxide, 160 mM at pH 10.5. Experimental conditions: 50 µm i.d. fused-silica capillary, 100 cm detector and total length, 20 kV, 7 s of hydrodynamic injection at 0.5 psi; sheath liquid, 2-propanol/water, 50:50 v/v, containing 0.1% TEA; flow rate, 3 µL/min; dry gas, 4 L/min, 150 °C; nebulizing gas pressure, 6 psi. MS analyses were carried out using negative polarity. Compound stability was 25% MS scan m/z 300−700 (target mass m/z 550). Sample was oxidized hop pellets of the variety Saaz. Figure 2. Base peak electropherogram and extracted ion electropherogram. Conditions: buffer ammonium carbonate/ammonium hydroxide, 160 mM at pH 9; 50 µm i.d. fused-silica capillary, 100 cm detector and total length, 20 kV, 7 s of hydrodynamic injection at 0.5 psi; sheath liquid, 2-propanol/water, 50:50 v/v, containing 0.1% TEA; flow rate, 3 µL/min; dry gas, 4 L/min, 150 °C; nebulizing gas pressure, 6 psi. MS analyses were carried out using negative polarity. Compound stability was 25% Figure 3. (a) Conversion of humulone into humulinone; (b) oxidation mechanism for the formation of the compound with molecular weight (M) 394 from humulinone; (c) conversion of lupulone into hulupone. Figure 4. Differences among samples of Saaz hops: (a) without oxidation; (b) with forced oxidation; (c) naturally oxidized. The separation conditions are shown in Figure 2 Figure 5. Differences among samples of Nugget hops: (a) without oxidation; (b) with forced oxidation; (c) naturally oxidized. The separation conditions are shown in Figure 2. Figure 6. Differences among samples of Magnum hops: (a) without oxidation; (b) with forced oxidation. The separation conditions are shown in Figure 2. Figure 7. Differences among samples of Columbus hops: (a) without oxidation; (b) with forced oxidation. The separation conditions are shown in Figure 2. Figure 8. (a) Electropherogram of a mixture of three trans iso-α-acid standards: 1, trans-iso-humulone and trans-iso-adhumulone; 2, iso-cohumulone. (b) Mass spectra of isohumulone and iso-adhumulone with a m/z ratio of 361.2. (c) Mass spectra of iso-cohumulone with a m/z ratio of 347.2. Table 1. Structures of compounds found in different varieties of hops without oxidation. [M-H]a analyte Saaz Nugget Magnum Columbus posthumulone 303.5 cohulupone 317.2 317.2 317.2 hulupone/adhulupone 331.2 331.2 331.2 cohumulone 347.3 347.3 347.3 347.3 humulone/adhumulone 361.2 361.2 361.2 361.2 cohumulinone 363.2 363.2 prehumulone 375.2 375.2 humulinone/adhumulinone 377.3 377.3 377.3 377.3 colupulone 399.3 399.3 399.3 399.3 lupulone/adlupulone 413.3 413.3 413.3 413.3 a[M-H] is the deprotonated ion. . Table 2. Structures of compounds found in different varieties of hops with forced oxidation [M-H]a analyte Saaz Nugget Magnum Columbus posthumulone cohulupone 317.2 317.2 317.2 317.2 hulupone/adhulupone 331.2 331.2 331.2 331.2 cohumulone 347.3 347.3 347.3 humulone/adhumulone 361.2 361.2 361.2 cohumulinone 363.2 363.2 363.2 363.2 prehumulone 375.2 375.2 humulinone/adhumulinone 377.3 377.3 377.3 377.3 colupulone 399.3 399.3 lupulone/adlupulone 413.2 413.2 413.2 a[M-H] is the deprotonated ion. Table 3. Structures of compounds found in naturally oxidized Saaz and Nugget hops [M-H]a analyte Saaz Nugget posthumulone cohulupone 317.2 317.2 hulupone/adhulupone 331.2 331.2 cohumulone 347.3 347.3 humulone/adhumulone cohumulinone 363.2 363.2 prehumulone humulinone/adhumulinone 377.3 377.3 colupulone lupulone/adlupulone a[M-H] is the deprotonated ion. Figure 1. Figure 2. Figure 3. Figure 4. Figure 5. Figure 6.