Application Note 1020 and Bitter Acids in by HPLC with UV and Electrochemical Detection

Paul A. Ullucci, David Thomas, and Ian N. Acworth Thermo Fisher Scientific, Chelmsford, MA

Key Words , bitter acids, xanthohumols, electrochemical array detection, differentiation, stability

Goal To develop gradient HPLC methods using a spectro-electro array platform to either measure specific analytes in beer samples or in a metabolomic approach to distinguish between different beer samples, as well as study beer stability.

Introduction The bitter acid method was used to study beer stability. A Beer is the most widely consumed alcoholic beverage in metabolomic approach was also developed where patterns the world and the third most popular drink after water of both known and unknown analytes were used to study and tea. It is typically brewed from four basic ingredients: differences between beer samples–an approach that is water, a starch source such as malted barley, brewer’s relevant to quality control. yeast, and a flavoring agent such as . Many varieties Experimental of beer result from differences in these ingredients, the Methods were developed for the measurement of either additives used and the brewing process followed. polyphenols or xanthohumols and bitter acids. Hops are the female flower clusters of a hop species, Standard Preparation Humulus lupulus. They are used as a flavoring and For the method, standards were prepared in stability agent in beer, for various purposes in other ethanol, methanol, or methanol/water solutions, depending beverages, and as an herbal medicine. Hops contain a upon solubility, at 100 or 1000 µg/mL. Working standards number of important including were prepared at 0.20, 0.50, and 1.0 µg/mL in 10% methanol (a prenylated ) and alpha- and containing 0.2% ascorbic acid/0.02% EDTA. beta-acids. As part of the beer brewing process, hops or hop extracts are added during the boiling of the wort. The For the bitter acids method, standard mixes were obtained alpha-acids (humulone, cohumulone, and adhumulone) from the American Society of Brewing Chemists: are slowly isomerized into the more soluble iso-acids, the International Calibration Standards for the HPLC Analyses main bittering substances in beer. Unfortunately, alpha- of Isomerized alpha-acids including: DCHA-Iso, ICS-12; acids can react with riboflavin and light to produce DCHA-Rho, ICS-R2; Tetra, ICS-T2; and DEHA-Hexa, compounds that give beer an off or skunky taste and ICS-H1. All acids were obtained as their dicyclohexylamine smell. Beta-acids (lupulone, colupulone, and adlupulone) salt. Stock standard solutions were prepared by dissolving do not isomerize during boiling and do not impart 100 mg of the standard material with 100 mL of acidified bitterness initially. However, during fermentation and methanol (0.1% phosphoric acid in methanol containing storage, beta-acids slowly create bitterness through (10 mg/L) as preservative and antioxidant) and oxidation affecting the long-term character of aged . sonicating for 15 minutes. Calibration standards were Furthermore, some secondary metabolites contribute to prepared in 50% acetonitrile containing 0.1% phosphoric the degradation of beer during storage with the formation acid and gallic acid (10 mg/L) in the range of 0.10−2.0 µg/mL. of haze (e.g., and their polymers, the ). In this study, two targeted assays were developed for the measurement of either polyphenols (including catechins and proanthocyanidins) or xanthohumols and bitter acids. 2 Sample Preparation Data Analysis Various beer samples were obtained from the local liquor Data were analyzed using Thermo Scientific Dionex store, including domestic beer and a light equivalent, Chromeleon Chromatography Data System 6.8 (SR 9) numerous domestic microbrews, European examples and CoulArray™ software 3.1. EC-array data were (Bavaria and Belgium), and a domestic extremely bitter transferred to Pirouette® software for chemometric (highly-hopped) beer. Beer samples were treated as analysis (Infometrix Inc., Bothell, WA) using a CoulArray follows: 0.50 mL of beer + 0.50 mL of acidified version 2.0 software utility (Pattern Recognition Setup acetonitrile (0.1% phosphoric acid and gallic acid (10 mg/L) Wizard). UV data were tabularized prior to transfer to in acetonitrile) were mixed, centrifuged, and the clear Pirouette. supernatant analyzed. For the stability study, beer samples Results and Discussion were transferred to a sealed container and kept at 4 °C in The spectro-electro array makes use of both the dark, and processed as needed. spectrophotometric and electrochemical data (EC).1 While Liquid Chromatography Method 1: Polyphenols UV data provides identification and quantitation of the major components in a sample, EC array detection Pump: Thermo Scientific Dionex LPG-3400BM with provides additional information. First, the EC array is SR-3000 solvent rack incredibly sensitive with low picogram limits of detection Autosampler: Thermo Scientific Dionex WPS-3000TBSL (LOD), and it is capable of measuring compounds missed UV Detector: Thermo Scientific Dionex DAD-3000RS by UV. Second, it voltammatrically resolves compounds diode-array detector that co-elute chromatographically. Third, the EC array is Channel 1: 218 nm Channel 2: 240 nm fully gradient compatible, thereby extending the number Channel 3: 254 nm Channel 4: 275 nm of analytes that can be measured in a sample. Fourth, the redox behavior of a compound reacting across the array EC Detector: Thermo Scientific Dionex CoulArray detector provides qualitative information and can be used for with thermal organizer analyte identification/authentication. EC Parameters: 16 channel array from 0 to +900 mV in +60 mV increments Polyphenol Method – Targeted Analysis The analytical figures of merit for this assay were Column: Thermo Scientific Acclaim 120, 1 C18 (3.0 × 150 mm, 3 µm particle size) described previously. Briefly, the limits of detection were typically 10−50 pg on column by ECD and 100−500 pg by Flow Rate: 0.65 mL/min UV. The limits of quantification were 200−1000 pg on Injection: 10 or 20 µL column by ECD and 500−5000 pg by UV. The response Mobile Phase A: 20 mM monobasic sodium phosphate, range was over seven orders of magnitude by ECD and five 3% acetonitrile, 0.2% tetrahydrofuran, pH 3.35 by UV. Typical R2 values were ~0.99 or better for all Mobile Phase B: 20 mM monobasic sodium phosphate, compounds. Average intra-day retention time precision for 50% acetonitrile, 10% tetrahydrofuran, pH 3.45 all analytes averaged 0.55% RSD over a 10 day period, with a range of 0.30%−1.22%. Examples of multi-channel EC Mobile Phase C: 90% methanol array chromatograms for two different beer samples are Gradient: 0-2 min: 2%B/3%C, 30 min: 97%B/3%C, presented in Figure 1A for high-hops beer and 1B for regular Concave, 45 min: 97%B/3%C domestic beer. The high-hops beer contains a great abundance of analytes, as confirmed in Table 1. Analyte Liquid Chromatography Method 2: Bitter Acids levels are in agreement with previously published data.

Column: Acclaim™ 120, C18 (3.0 × 150 mm, 3 µm A A particle size) 2.50

Flow Rate: 0.65 mL/min 2.00 Injection: 20 µL 1.50 Mobile Phase A: 25 mM sodium perchlorate, 50% acetonitrile, 1.00 2.5 mM perchloric acid 0.50 0.00 Mobile Phase B: 25 mM sodium perchlorate, 90% acetonitrile, 0.00 10.00 20.00 30.00 40.00 min 2.5 mM perchloric acid A B Mobile Phase C: 90% methanol 2.50 Gradient: 0-3 min: 0%B/3%C, 30 min: 40%B/3%C, 2.00 40 min: 97%B/3%C, 45 min: 97%B/3%C 1.50 EC Parameters: Thermo Scientific Dionex model 5011A dual 1.00 channel coulometric electrochemical cell 0.50 E1: +550 mV 0.00 0.00 10.00 20.00 30.00 40.00 min E2: +850 mV Figure 1. Polyphenol method chromatograms of (A) high-hops beer and B) regular domestic beer Table 1. Polyphenol data presented in mg/L. Beer 1 = high-hops; measured in each sample. Principal component analysis 3 Beer 3 = regular (PCA) was then used to differentiate samples for both EC data (Figure 2A) and UV data (Figure 2B). The EC data Compound Beer 1 Beer 3 gave the best differentiation between samples and could Hydroxybenzyl alcohol 2.32 ND distinguish between the light and normal beer, Irish stout, 2-Hydroxybenzyl alcohol 0.04 ND Belgian beer, and the extra hopped IPA. UV data were less 3,4- 2.10 ND selective with no ability to distinguish between light and 4-Hydroxybenzaldehyde 0.16 ND normal beers. 4-Hydroxybenzoic acid 0.38 0.36 4-Hydroxyphenyl acetic acid 0.14 0.24 Beer 7 A Factor2 Ultra 4-Hydroxycoumarin 11 0.46 IPA 0.2 0.02 Factor1 0.14 0.1 Beer 13 Beer 8 USA Full and Carnosol 0.38 ND USA Microbrews Light Beer 5 4.48 1.8 BFaeerctor 123 Beer 10 Beer 9 1.2 0.25 Beer 6 Irish 0.52 0.04 Stout 0.12 0.26 Belgian Beer 11 Epicatechin 1.76 0.40 Epicatechingallate 1.54 0.26 Epigallocatechin 0.14 0.16 Factor2 B BEER 10 1.38 0.64 Irish Ethyl 0.2 0.02 Stout 1.42 2.54 Factor1

Gentisic acid 0.24 0.06 BEER 8 USA Full, BEER 9 BEER 11 Factor3 BEER 5 0.40 0.04 USA Light, BEER 12 Bavarian, BEER 13 Isorhamnetin 0.94 ND Belgian Microbrews, Ultra IPA Isoxanthohumol 1.16 0.14 BEER 6 1.04 ND BEER 7 Myricetin 0.5 0.2 2.08 5.1 Figure 2. Principal component plots for (A) ECD and (B) UV Data p-Coumaric acid 2.02 2.8 1.56 ND Bitter Acid Method – Targeted Analysis 0.18 0.04 The bitter acid method showed similar figures of merit to Sinapic acid 0.68 0.58 the polyphenol method above. All calibration curves Syringaldehyde 1.39 ND showed good linear regression (R2 > 0.996). Peak area RSDs over a twenty-hour run were as follows: 0.08 0.1 isoxanthohumol and xanthohumol, 1.2%; alpha and beta Thymol 0.78 0.09 bitter acids, 2.5%; and trans- and cis-iso-alpha acids, Umbelliferone 0.34 0.64 2.4%. Limits of detection for most analytes were in the Vanillic acid 0.22 0.15 picogram (on-column) range. Rather than using an EC Vanillin 1.06 0.26 array approach, a simple two-channel cell was used to detect xanthohumol and the alpha- and beta-bitter acids Xanthohumol 0.26 0.04 on the first lower potential channel, and isoxanthohumol,

and the cis- and trans-iso bitter acids on the second higher Polyphenol Method – Metabolomic Study potential channel. Example chromatograms are shown in A simple metabolomics experiment was conducted to Figure 3A (standards) and Figure 3B (extra hopped IPA). evaluate whether the spectro-electro array platform could As expected, the Ultra IPA beers were abundant in the be used to differentiate between different beer types bitter acids and related compounds, more so than including: matched domestic normal and light beers, a matched normal and light beers (Table 2). variety of domestic microbrews, two European beers (from Bavaria and Belgium), an Irish stout, and a domestic Ultra (extra hopped) IPA. Metabolomic profiles containing several hundred analytes, including both known (Table 1) and unknown compounds, were Application Note 1020 - 2 WEEK  1 WEEK +847 295 7500 48 +41 62 205 9966 24 +46 8 473 3380 , 169-178. 169-178. 106, +886 2 8751 6655 0 +44 1276 691722 Sweden Switzerland Taiwan UK USA and Canada 2000, 



9 8 7 6 5 4 3 2 1 0

10 Concentration (mg/L) Concentration Alteration of H. G.; Overmeire, Aerts, L.; De Cooman, Ageing, the Profiles of Iso-alpha-Acids During Beer Marked Instability of trans-Iso-alpha-Acids and in Relation Consistency Implications for Beer Bitterness, Journal of the Institute of to Tetrahydroiso-alpha-acids, Brewing, Ullucci, P.A.; Bailey, B.; Acworth, I.N.; Crafts, C.; I.N.; Crafts, Acworth, B.; Bailey, P.A.; Ullucci, A Novel Platform Array: The Spectro-Electro M. Plante, for the Measurement of Secondary Metabolites in Part 1. and Beverages. Food, Supplements, Botanicals, 2012, Pittsburgh Conference, Theory and Concepts, Poster 1770-1 P. The polyphenol method can be used in a targetedThe polyphenol method approach to accurately and sensitively measure and polyphenols in phenolic acids, numerous phenols, beer and other samples not possible by methods employing UV detection alone. of numer Metabolomic approaches using the patterns used toous known and unknown analytes can be Such an differentiate between different samples. product approach can be used to study fermentation, and authenticity. stability, targetedThe bitter acid method enabled the sensitive xanthohumol, measurement of isoxanthohumol, the trans- and cis-iso-alpha bitter prenylnaringinen, in a single run. acids and the alpha and beta bitter acids for solid phaseUse of EC detection eliminated the need extraction procedures for sample pre-concentration This commonly used in UV detection methods. over a twoapproach was used to measure beer stability week period.

2. References References 1. Figure 4. Stability data for Ultra IPA beer (Beer 1) Conclusion • • •

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lupulone lupulone

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B A

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colupulone colupulone +39 02 51 62 1267 ND ND ND ND ND 1.6 2.6 0.02 0.28 Beer Light

40.00 Beer 4

40.00 Ireland Italy Japan Korea Singapore

humulone

adhumulone/humulone & adhumulone

cohumulone

cohumulone ND ND ND ND 3.8 3.2 0.03 0.38 0.20

Beer 3

trans-iso-adhumulone trans-iso-adhumulone Regular

trans-iso-adhumulone trans-iso-adhumulone -rho-isohumulone, 30.00

30.00

trans-iso-humulone trans-iso-humulone cis trans-iso-humulone

3 iso cis

3 iso cis

2 iso cis

cis iso 2 2 iso cis 7.0 7.6 1.3 0.4 9.0 6.4 IPA 11.0 12.2 0.48 Ultra Beer 2

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trans-iso-cohumulone

cis-iso 1 1 cis-iso +33 1 39 30 01 10

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xanthohumol

20.00

20.00 xanthohumol China India Denmark France Germany 8.4 9.2 6.8 IPA 19.1 2.10 12.8 10.6 0.52 0.90 Ultra Beer 1

-rho-isocohumulone,

prenylnaringinen prenylnaringinen

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10.00 prenylnaringinen

isoxanthohumol isoxanthohumol

isoxanthohumol +31 20 683 9768 +32 3 353 42 94 +61 3 9757 4486

-rho-isoadhumulone. Absolute identification possiblewas not  iso-humulone iso-adhumulone iso-cohumulone -isomers were cis +43 1 616 51 25 +55 11 3731 5140 iso-acid 1 iso-acid 2 0.00

cis cis 0.00 2.0 1.0 0.0 5.0 4.0 3.0 A A 2.0 1.0 0.0 5.0 4.0 3.0 Adhumulone/Humulone Compound Cohumulone Isoxanthohumol Xanthohumol trans- trans- cis- trans- cis-

AN70162_E 07/16C Brazil Australia Austria Benelux (-55%). These data are consistent with previous studies. (-55%). iso-cohumulone and trans- trans-iso-adhumulone (-56%), are the property of Thermo Fisher Scientific Inc. and its subsidiaries. This informationcapabilities is presented as of Thermoan example Fisherof the Scientific Inc. products. It is not intended to encourage use thatof these might products infringe the in any intellectual manners property rights of others. Specifications, details. for representative sales local your consult Please countries. all terms in and available pricing are products are subject to change. Not all iso-humulone (-67%), trans-iso-humulone (-67%), particularly for cis-iso 2 (61%), www.thermofisher.com/dionex ©2016 Thermo Fisher Scientific Inc. All rights reserved. Pirouette is the trademark of Infometrix, Inc. All other trademarks Figure 4. Marked decreases in many analytes were seen but Marked decreases in many analytes Figure 4. sample tested over a two week period are presented in Data for the stability of a variety of analytes in an Ultra IPA Data for the stability of a variety of analytes in an Ultra IPA was used to evaluate the stability of a selection of the beers. was used to evaluate the stability of a selection of the beers. decrease of beer bitterness. The current bitter acids method decrease of beer bitterness. degradation of iso-alpha-acids is the cause of the gradual flavor qualities of beer during aging. It is believed that It flavor qualities of beer during aging. plays an important role in the deterioration of taste andplays an important role in the deterioration Auto-oxidation, including decomposition of iso-alpha-acids, including decomposition of iso-alpha-acids, Auto-oxidation, Beer StabilityBeer Study Figure Chromatograms 3. of (A) bitter acids standard mixture; The (B) Ultra IPA beer sample. Blue trace green mV; at 550 trace at mV. 850 Table 2.Table Hops bitter acids data presentedin mg/L due to the lack of single standards. and