Journal of the American Society of Brewing Chemists The Science of Beer

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Hop Aroma and Hoppy Beer Flavor: Chemical Backgrounds and Analytical Tools—A Review

Nils Rettberg, Martin Biendl & Leif-Alexander Garbe

To cite this article: Nils Rettberg, Martin Biendl & Leif-Alexander Garbe (2018) Hop Aroma and Hoppy Beer Flavor: Chemical Backgrounds and Analytical Tools—A Review , Journal of the American Society of Brewing Chemists, 76:1, 1-20 To link to this article: https://doi.org/10.1080/03610470.2017.1402574

Published online: 27 Feb 2018.

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Full Terms & Conditions of access and use can be found at http://www.tandfonline.com/action/journalInformation?journalCode=ujbc20 JOURNAL OF THE AMERICAN SOCIETY OF BREWING CHEMISTS 2018, VOL. 76, NO. 1, 1–20 https://doi.org/10.1080/03610470.2017.1402574

Hop Aroma and Hoppy Beer Flavor: Chemical Backgrounds and Analytical Tools— A Review

Nils Rettberga, Martin Biendlb, and Leif-Alexander Garbec aVersuchs– und Lehranstalt fur€ Brauerei in Berlin (VLB) e.V., Research Institute for Beer and Beverage Analysis, Berlin, Deutschland/Germany; bHHV Hallertauer Hopfenveredelungsgesellschaft m.b.H., Mainburg, Germany; cHochschule Neubrandenburg, Fachbereich Agrarwirtschaft und Lebensmittelwissenschaften, Neubrandenburg, Germany

ABSTRACT KEYWORDS Hops are the most complex and costly raw material used in brewing. Their chemical composition depends Aroma; analysis; beer flavor; on genetically controlled factors that essentially distinguish hop varieties and is influenced by environmental gas chromatography; hops factors and post-harvest processing. The volatile fingerprint of hopped beer relates to the quantity and quality of the hop dosage and timing of hop addition, as well as the overall brewing technology applied. Analytically, the aroma of hops and the flavor of hoppy beers cannot be measured by quantification of a single odorant; moreover, the selection of several key compounds or a comprehensive characterization (profiling) seems reasonable. Analysis of hops and beer is challenging. The selective enrichment of volatiles from complex matrices, separation, unambiguous identification, and precise quantification are the keywords used in this context. This review outlines the synthesis of relevant hop aroma compounds within the plant. The process that incorporates hops into the final beer is described using the hopping techniques used in the industry. Due to the complexity and multiplicity of chemical compounds found in hops, an attempt was made to simplify the information presented by separating the chemical compounds considered into two broad groups: terpenoids and nonterpenoids. This review summarizes approaches commonly used for analysis of hop aroma compounds in hops and beer.

Introduction Role of hops in modern brewing Hop derived volatiles and their transformation products strongly The volatiles of hops, collectively known as “hop oil,” are a very impact beer flavor. Extensive research has been performed to heterogeneous and complex mixture of hundreds of com- unravel the secrets of hop aroma by analyzing its chemical com- pounds. Gravimetrically, the hop oil represents 0.5–3.0% (by position and by identifying the high impact odorants of hop mass) of the hop cone dry matter,[22,23] in which relatively few, – essential oil.[1 5] Other papers focus on unlocking the secrets of but quantitatively important compounds, namely the terpene hoppy beer flavor by elaborately studying factors that influence hydrocarbons and terpenoids, are directly biosynthesized (cf. effective enrichment, as well as losses of hop volatiles during beer 1.3). The vast majority of volatiles are either by-products of – production.[6 9] In contrast to the seemingly well-understood plant metabolism or they evolve from (oxidative) secondary principles of beer bittering, chemistry of transfer, stability, repro- reactions of volatile and nonvolatile precursor molecules. The ducibility, and metabolism of hop aroma compounds in beer is combination of terpene biosynthesis and secondary reactions – – still rather fragmentary.[10 13] Several research groups[9,14,15 19] leads to the fact that hop oil composition importantly depends have identified numerous compounds that contribute to hoppy on genetic (cultivar) and cultural factors (ripeness, geography), beer flavor, and they mostly agree that linalool, geraniol, b-dam- but is also liable to considerable changes during ripening, kiln- – ascenone, b-citronellol, esters (e.g., ethyl 4-methylpentanoate and ing, processing, and, finally, hop storage.[24 29] The secondary (Z)-4-decenoate), and organic acids (2- and 3-methylbutanoic reactions, such as oxidation and hydrolysis, continue through- acid) are significant contributors to hoppy beer flavor. However, out the brewing process. As a result, only a few hop-derived most publications either introduce new, so-called high impact, odorants survive the brewing process in their original odorants or deny the relevance of other previously published sub- state.[30,31] The occurrence of either bitter or aromatic charac- stances. Obviously, hoppy beer flavor is influenced by the sheer teristics in beer depends on numerous factors relating to the number of possible chemical combinations of substances, as well hops, namely their chemical composition, the amount added, as by synergistic and masking effects among volatile and nonvol- the type of hop product, and the time of hop dosage.[32] Con- atile beer constituents.[20,21] Thus, early attempts to create stan- ventionally, beer bitterness is achieved by adding hops to the dard measures for hop aroma (hop aroma units) failed.[3] From hot wort at the beginning of the kettle boil (kettle hopping). today’s view, it seems difficult to compile a brief and universal The primary reason for an “as early as possible” hop addition is list of chemicals that allows a prediction of the aroma impact of the thermal conversion of hop bitter acids (a-acids) into bitter hops on a finished beer. tasting and water soluble iso-a-acids; whereas, the yield of iso-

CONTACT Nils Rettberg [email protected] © American Society of Brewing Chemists, Inc. 2 N. RETTBERG ET AL. a-acids increases with boiling time, and the majority of volatiles compounds represent up to 80% of the total volatiles of hop is lost by evaporation.[6,30,31,33] Beers with hop aroma are com- cultivars bred for brewing. In some aroma hop varieties such as monly achieved by adding multiple and late hop dosages. In Saazer and Tettnanger, b-farnesene, a noncyclic sesquiterpene, order to achieve this, hops are added toward the end of kettle- is present in relevant concentrations (100–1500 mg/kg). In boil or to the whirlpool (late hopping) to green and bright beer addition, hops contain numerous bicyclic and tricyclic minor (dry hopping). The aroma profiles of late and dry hopped beers terpene hydrocarbons (i.e., bergamotene, aromadendrene, a- – – differ considerably.[34 36] In late hopping, some hop derived and b-selinene, germacene, and amorphene).[51 53] Terpene volatiles evaporate, while some undergo thermal conversion hydrocarbons are accompanied by a vast number of terpenoids. and oxidation.[34,37,38] Late hopped beers contain only trace lev- Terpenoids are terpene carbon skeletons that contain func- els of monoterpene and sesquiterpene hydrocarbons but con- tional groups. Terpene hydrocarbons and terpenoids play mul- tain relevant concentrations of polar oxygenated terpene tiple roles in plant physiology. Most importantly, they are derivates (e.g., humulene epoxides or linalool oxides), ethers, synthesized and emitted to defend the hop plant against herbi- hop derived ketones, and esters.[31] Flavor descriptors used for vores and pathogens and to attract pollinators and seed dissem- – late hopped beers include spicy, noble, herbal, woody, and, to inators.[54 56] Terpene biosynthesis primarily occurs in the some extent, estery and fruity,[22] while citrus-like, floral, and lupulin glands (glandular trichomes) of the cone. It is a tightly pine-like are used for dry hopped beers. Dry hopping is not regulated reaction cascade, using isopentenyl diphosphate and confined to an exact recipe. It is a synonym for a broad range dimethylallyl diphosphate as universal building blocks. Both of of techniques, in which hops are added to cold beer. Cones and these compounds arise from either acetyl-CoA formed by the pellets are either added to primary or secondary fermentation, cytosolic mevalonic-acid pathway or from pyruvate and in-line before filtration, or even to the cask.[39,40] Dry hopping glyceraldehyde-3-phosphate formed by the plastidial 2-C- – is carried out in static or dynamic systems, meaning that circu- methylerythritol 4-phosphate pathway.[57 60] In monoterpene lation, mixing, or agitation might or might not be performed. biosynthesis, prenyltransferases catalyze the condensation of Chemically speaking, dry hopping is a cold extraction that uses isopentenyl diphosphate and dimethylallyl diphosphate into an aqueous ethanol solution as solvent. Disregarding some geranyl pyrophosphate or neryl diphosphate. Geranyl pyro- green hop beers that are produced by adding freshly harvested phosphate is the parent of many noncyclic monoterpenes (e.g., hop cones, hop pellets or powders are used for dry hopping. myrcene), while neryl diphosphate is the key precursor of cyclic Both have the advantage that they contain mechanically terpenes (e.g., limonene). In sesquiterpene synthesis, the con- crushed lupulin glands, which facilitate the extraction of bitter- densation of isopentenyl diphosphate and two dimethylallyl ness and aroma from the beer.[39] The ability to extract volatiles diphosphate molecules gives rise to farnesyl pyrophosphate. (and nonvolatiles) from hops into beer is strongly influenced Elimination of pyrophosphoric acid from farnesyl pyrophos- by their respective chemical structure: Polar compounds such phate results in b-farnesene, while additional cyclization as linalool can be quantitatively transferred, whereas nonpolar of pyrophosphoric acid gives rise to a-humulene and b-caryo- hydrocarbons such as myrcene are hardly recovered due to phyllene. Minor mono- and sesquiterpenes, and also important their limited solubility, adsorption onto yeast cells, or stripping terpenoids such as geraniol and linalool, are finally generated losses with carbon dioxide.[7,41] In the case where hops are by a large family of enzymes known as terpene synthases/ added to beer containing viable yeast cells (active yeast cyclases.[61,62] enzymes), extraction is accompanied by biotransformation of – some compounds.[42 44] The action of yeast on volatiles or their Fate of a-humulene, b-caryophyllene, and myrcene during nonvolatile precursors (i.e., glycosides) produces a variety of hop processing and brewing compounds contributing to hop character in beer. The main biotransformations of hop derived aroma compounds in fer- Terpene hydrocarbons such as myrcene, a-humulene, and mentations with Saccharomyces cerevisiae are the reduction of b-caryophyllene are hydrophobic. Their isoprene body and the – carbonyl compounds or ethers into alcohols and diols,[45 47] absence of polar functional groups cause their limited solubility – isomerization of monoterpene alcohols,[42 44] release of glyco- in aqueous solutions such as wort and beer. Even though myr- – sidically bound aroma precursors,[48 50] trans-esterification, or cene, a-humulene, and b-caryophyllene are the principal vola- hydrolysis of esters,[18] as well as the conversion of acids into tiles of hops, they are rarely found in kettle and late hopped (ethyl) esters.[15,16,18] In a nutshell, the complex composition of beers.[44,46] During beer production, considerable losses occur the hop oil and disproportionate extraction of volatile and non- by evaporation that are washed out by carbon dioxide in the volatile compounds, as well as multiple chemical and biochemi- fermentation tank and adsorption onto the biomass.[16,41] Gen- cal conversion reactions, challenge brewers and brewing erally speaking, terpene hydrocarbons do not contribute to research. Hopping is a complex technology, and there remain beer flavor, except in dry hopped beers.[14,34] Nevertheless, their many technological and analytical aspects that are not fully oxidation during hop storage and wort boiling gives rise to – understood. numerous flavor active, oxygenated derivates.[46,63 65] Humu- lene oxidation theoretically results in the formation of three humulene monoepoxides (numbered I–III), 12 diepoxides, and Biosynthesis of terpenes and terpenoids 18 triepoxides. Only mono- and diepoxides were found in beer The main constituents of fresh hop essential oil are monoter- and hops;[46,66] refer to Peacock and Deinzer[67] for a detailed penes (C10 body) and sesquiterpenes (C15 body), namely description of humulene epoxide stereochemistry. During fer- myrcene, a-humulene, and b-caryophyllene. These three mentation and beer storage, proton catalyzed hydrolysis of JOURNAL OF THE AMERICAN SOCIETY OF BREWING CHEMISTS 3

Figure 1. Chemical structures of a-humulene and oxygenated humulene derivates. Chiral centers are not indicated.

Figure 2. Chemical structures of b-caryophyllene, caryophyllene oxide, and 14-hydroxy-b-caryophyllene. Chiral centers are not indicated. humulene epoxides produces humulenol II and humulol as aroma.[30,46,63,73,74] Two explanations seem reasonable: either the main products.[28,31] The structure of a-humulene and sub-threshold perception of these substances results from addi- selected oxygenated humulene derivates is shown in Figure 1. tive effects with other hop derived odorants[75] or the afore- Individually, the odors of humulene epoxides, humulol, and mentioned compounds are accompanied by related, but thus humulenol II are described as hay-like, moldy, cedar, and sage- far undefined, molecules.[8,23,76] brush; humuladienone as flowery and fresh. a-Humulene and Myrcene (cf. Figure 3) is the principal volatile of most hop b-caryophyllene are both said to have a spicy and woody varieties. Myrcene’s concentration ranges between 3 and 10 odor.[3,22,63] The oxidation of b-caryophyllene predominantly mg/g hop dry matter and its odor is described as resin-, pine-, yields caryophyllene oxide (musty, floral, spicy, cedar smelling) and herb-like. Myrcene is a significant contributor to fresh hop and 14-hydroxy-b-caryophyllene (woody-cedarwood odor), aroma;[4,77] yet, its concentration decreases strongly during aer- but also caryolan-1-ol (gasoline, fruity, lemon odor), 4S-dihy- obic hop storage and, more importantly, during the brewing drocaryophyllene-5-one, (3Z)-caryophylla-3,8(13)-diene-5a-ol, process. During drying and aerobic hop storage, myrcene – and caryophylla-4(12),8(13)-diene-5a/b-ol.[23,67 71] The struc- mainly evaporates, although a minor amount of polymerization tures of b-caryophyllene and its major oxidation reaction prod- and oxidation occurs. The autoxidation of myrcene gives rise to ucts are shown in Figure 2. The contribution of oxygenated multiple cyclic reaction products (e.g., a-pinene, b-pinene, sesquiterpenes to beer flavor is not yet fully understood. camphene, p-cymene) and also forms terpenoids such as linal- Their flavor threshold values are generally much higher than ool, nerol, geraniol, citral, a-terpineol, or carvone.[78] Oxygen- their levels in beer,[68,72] but their concentration does, in ated myrcene derivates can be found in beer, but myrcene itself fact, correlate with the so-called noble or kettle hop is effectively evaporated during wort boiling, lost by scrubbing 4 N. RETTBERG ET AL.

Figure 3. Chemical structures of myrcene and prominent myrcene oxidation products.

Figure 4. Chemical structures of geraniol, geranyl acetate, and geranyl-b-D-glucoside. during fermentation, or adsorption onto yeast.[41,43,76] Myrcene present in the bound form, they were accompanied by concentrations in beer in the range of its flavor threshold (30– multiple aliphatic alcohols (e.g., (Z)-3-hexen-1-ol), aromatic 100 mg/L) are only obtained by dry hopping. structures (e.g., benzyl alcohol), and norisoprenoids. Obviously, there are strong differences in the concentration levels of glycosidically bound odorants among hop varieties. Terpenoids According to Wilhelm,[84] there is a correlation between the Terpenoids are terpene carbon skeletons that primarily con- concentrations of free and bound linalool, where bound linal- tain hydroxyl, carboxyl, ester, and ether functional groups. ool contributes to 21–36% of the total linalool concentration There are considerable differences in the qualitative and (the sum of free and bound linalool). Thermal, enzymatic, quantitative composition of the terpenoid fractions among and acid catalyzed cleavage release aglycones during beer pro- – hop varieties. These differences not only depend on hop vari- duction, thus increasing the levels of odorants in beer.[48 50] ety but are also susceptible to cultivation factors (climate, soil, Same accounts for esterified terpene alcohols (mainly gera- fertilizers, age of the hop plant).[79] The concentration of niol), which are abundant in certain hop varieties (e.g., Cas- terpenoids increases during ripening of the hop cone,[32,80] cade and Polaris) but hardly found in beer.[81,85] From an – hop processing, and (aerobic) storage.[24 28] From the brewers analytical point of view, the major difference between esteri- viewpoint, the most relevant terpenoids in hops are the fied- and glycosidically-bound odorants is their volatility. monoterpene alcohols, linalool, geraniol, and also their iso- Whereas volatile esters can be monitored in a conventional mers nerol and a-terpineol.[12,15,30] In hops, those terpene hop oil analysis, nonvolatile glycosides require alternative, alcohols occur as free volatiles but are also esterified[81] and generally more elaborate, analytical routes. Straightforward bound to carbohydrates (glycosides).[50,82] Figure 4 shows the gas chromatography–mass spectrometry (GC–MS) and liquid chemical structures of geraniol, geranyl acetate, and geranyl- chromatography–mass spectrometry (LC–MS) approaches b-D-glucoside. The principal esters present in hop oil are ger- for the analysis of glycosidically-bound odorants from hops anyl esters, namely geranyl acetate, geranyl propionate, and and hop products have been described by Ting et al.,[83] Koll- geranyl isobutyrate.[31,46,47] Glycosides are nonvolatile odor- mannsberger et al.,[50] and Wilhelm.[84] Interestingly, those less compounds built from a sugar-moiety (usually ß-D-glu- routes are (thus far) not widely used for estimating the brew- cose) and a nonsugar-moiety (e.g., a monoterpene alcohol) ing value of hops. In addition to the most relevant monoter- called aglycone. Ting et al.[83] found approximately 60 agly- pene alcohols, hops contain numerous sesquiterpene alcohols cones in hops, of which most were bound to glucose (92%) in such as farnesol, nerolidol, a-/g-eudesmol, and epi-cubenol, the C1 position (55%). The principal monoterpene alcohols or terpene aldehydes such as citral. Compared to the mono- found in a bound form were linalool and a-terpineol. When terpene alcohols these substances are usually found in much JOURNAL OF THE AMERICAN SOCIETY OF BREWING CHEMISTS 5

Figure 5. Structures of prominent (bi)cyclic ethers in hops and beer. Chiral centers are not indicated.

Figure 6. Chemical structures of the isomeric terpene alcohols linalool, geraniol, a-terpineol, and nerol as well as b-citronellol. Asterisks indicate chiral centers. lower concentrations.[31,46,47] For comprehensive compila- frequently found in hopped beers but not in hops. The chemi- tions of hop terpenoids, refer to Eyres and Dufour,[22] Tressl cal structures of the isomeric monoterpene alcohols and b-cit- et al.,[31] Nance et al.,[77] and Sharpe and Laws.[86] ronellol are shown in Figure 6. As indicated, linalool, Terpenoids can either be products of biosynthesis or sec- b-citronellol, and a-terpineol are chiral molecules occurring as ondary reactions. Biosynthetic formation is tightly regulated at (R)- and (S)-enantiomers. These compounds have remarkable the genetic level (transcription); their synthesis involves multi- aroma properties; they impart floral, geranium-like, fresh, and ple terpene synthases/cyclases.[61,62] Biosynthesis is the primary citrus notes to beer. They stand out as key components in hops source for qualitative and quantitative variations in the occur- and beer among the multiple hop derived volatiles.[76] Even rence of abundant terpenoids across hop cultivars.[79,87] The though these terpene alcohols possess the same molecular for- occurrence of many (minor) terpenoids in hop products also mula (except b-citronellol), they differ significantly not only in evolves from secondary reactions, such as air oxidation and their aroma impressions but also in their odor activity.[30,75] ester hydrolysis. There are numerous comprehensive papers (R)-Linalool, geraniol, and b-citronellol all have a flavor thresh- discussing the changes of the volatile fraction of hops during old of <10 mg/L in beer, whereas the threshold of nerol, a-ter- processing and storage.[4,46,47,51,88] It is generally agreed that pineol, and (S)-linalool is reported to be a factor of 10 to 50 the total concentration of volatiles decreases due to evaporation higher.[43,85,89] As previously described, the monoterpene alco- and autoxidation during hop storage, thereby shifting the ratio hols are effectively transferred from hops into wort and beer, between terpene hydrocarbons and oxygenated terpenoid vola- but they are also released from either volatile derivates (e.g., tiles. Qualitative and quantitative changes of the mono- and esters) or nonvolatile precursors (glycosides) during the brew- – sesquiterpenes during hop storage and beer production have ing process.[7,46,48 50] The liberation of bound monoterpene been previously described. Similar observations can be made alcohols is either a result of high temperatures during boiling, for the terpenoids: Evaporation, oxidation, and ester hydrolysis or due to acid or enzyme catalyzed hydrolysis. The metabolism increase the concentration of oxygenated low molecular weight of monoterpene alcohols during fermentation has been volatiles. Prominent products of terpenoid oxidation are described by numerous researchers; the works of King and (bi)cyclic ethers such as furan and pyran linalool oxides, kara- Dickinson[42,43] and also of Takoi et al.[44,89] are frequently hana ether, or hop ether (cf. Figure 5). These odorants exhibit cited. A fundamental genetic analysis of the geraniol metabo- spicy and woody, kettle hopped odors; their concentration lism upon fermentation was published by Steyer et al.[90] in hops and beer is in the low mg/kg and mg/L range, Researchers agree that geraniol is transformed into b-citronel- respectively.[22,31,63] lol and linalool, while linalool and nerol isomerize into a-ter- Upon fermentation, terpenoids undergo several (bio)trans- pineol during fermentation. While the reduction of geraniol formations. Most research has focused on the fate of the mono- into b-citronellol is clearly catalyzed by the OYE 2 enzyme,[90] terpene alcohols. Linalool and geraniol are the principal the transformation of geranyl acetate into citronellyl acetate monoterpene alcohols of hop oil and are frequently found in has been observed but not characterized in detail.[43,90] A com- hopped beers. They are usually accompanied by the isomers prehensive review on the biotransformation of hop-derived nerol and a-terpineol (molecular mass 154 g/mol), as well as aroma compounds was written by Praet et al.[21] Indeed, the b-citronellol (molecular mass 156 g/mol). b-Citronellol is compositional changes of monoterpene alcohols during 6 N. RETTBERG ET AL. fermentation are known, but there are reports from associated hottest current topics. Although terpenes and terpenoids are research fields that might be of relevance to brewers; some absolutely regarded as positive (beer) flavorings, most sulfur wine yeast strains are able to de-novo synthesize monoterpene compounds are detrimental to the quality of fresh beers.[17] alcohols in relevant concentrations.[91] It is also worth men- The human olfactory system is sensitive to sulfur compounds, tioning that linalool, nerol, geraniol, and a-terpineol undergo a and, thus, they tend to have low thresholds (ng/L or ppt).[22,106] variety of transformation reactions in dilute acidic media,[92,93] Depending on their chemical structure, they exhibit cheesy, which is possibly of relevance with regard to the stability of cooked vegetable-like, burnt, musty, and rubbery odors. How- hop flavor upon beer storage. ever, there are also some sulfanyl esters, sulfur containing alco- hols, and ketones that exhibit fruity, citrus, blackcurrant, peach, or muscat-like odors.[102] Thus far, the most relevant Norisoprenoids in hops and beer sulfur compound contributing (positively) to hoppy beer flavor Carotenoids are tetraterpenoids (8 isoprene molecules) con- is 4-mercapto-4-methylpentan-2-one (4MMP). It was shown taining 40 carbon atoms. They are important plant pigments. that 10 ng/L spiking was sufficient to emphasize the fresh and [107] The degradation of carotenoids releases flavor activated noriso- fruity aroma of lager beer. Its typical fruity blackcurrant fla- prenoids. Two norisoprenoids (C13), namely b-ionone and vor is a key characteristic of beers hopped with U.S. grown Cas- [102] b-damascenone, have been identified in malt, hop, and beer; cade, Simcoe, Summit, and Apollo. their chemical structures are shown in Figure 7. b-Damasce- none is perceived as a fruity and honey-like aroma, whereas b-ionone is shown to promote fruity floral/violet flavor in Formation and relevance of lipid and bitter acid beer.[16,17,19,33,37,63] b-damascenone is one of the primary odor- derived aroma components ants of pale lager beer, and its odor threshold was reported to Although terpenes and terpenoids are the major constituents of [19] be 4 ng/L in water. In hops, b-damascenone has been traced hop oil, many other trace substances complement the aromatic [17] in its volatile state; additionally, 3-hydroxy-b-damascone- mixture. The occurrence of sulfur compounds and norisopre- [50] glucoside has been identified. As is the case for the release of noids was previously briefly outlined. In addition, multiple non- terpene alcohols, the aglycone, here b-damascenone, can be terpenoid odorants exist that are also significant contributors to released by thermal or enzymatic hydrolysis of the sugar moi- the hop aroma such as aldehydes, ketones, alcohols, acids, lac- ety. It is agreed that the concentration of b-damascenone in tones, and esters. A comprehensive compilation of hop derived beer increases during long term storage or forced aging, respec- volatiles is given by Sharpe and Laws.[86] They listed approxi- [94,95] tively. A concise review of the fate of b-damascenone in mately 60 aldehydes and ketones, 70 esters, 50 alcohols, 25 acids, [96] beer is given by Sefton et al. and 30 oxygen containing heterocyclic compounds present in the hop oil. Indeed, the definite assignment of the origin of each specific compound is difficult. Generally, nonterpene volatiles in Sulfur compounds hop oil are by-products of central plant cell metabolisms. Mole- Hops contain various sulfur compounds. These include methyl cules including branched carbon skeletons (e.g., isobutyl isobu- thioesters,[97,98] thiophenes,[99] methyl- or polysulfides,[97,100] tyrate, isoamyl isobutyrate, and 3-methyl-1-butanol) and polyfunctional thiols,[101,102] and also sulfur adducts of myrcene aromatic structures result from amino acid biosynthesis and (myrcene disulfide), a-humulene, or b-caryophyllene.[99] The catabolism.[108] Aliphatic acids, their corresponding (methyl) complexity of sulfur containing compounds in hops and beer is esters, and also aldehydes are significant contributors to hop huge.[103] In some varieties, such as the New Zealand grown aroma and beer flavor. They originate from biosynthesis and Nelson Sauvin hops and particularly U.S. grown Cascade, Citra, breakdown of lipids and hop bitter acids.[29,44,47] Simcoe, and Tomahawk, polyfunctional thiols were reported as Lipids are a source of multiple potent odorants in numerous key contributors to the specific aroma of those varieties.[104,105] foods, beer, and hops. The oxidative degradation of (poly) Compared to the oxygenated compounds (thus far), a minimal unsaturated fatty acids produces a series of alkanals, alkenals, amount of research has been performed to study the relevance and alkadienals, saturated and unsaturated ketones, lactones, of sulfur compounds. Obviously, this is due to the complexity alcohols, and polyenes. Lipid degradation products are charac- of their analysis and extremely low concentration (ng/L), rather terized by extremely low sensory thresholds; humans easily than their insignificance. The work in this field of aroma detect them in ng/L concentrations by smelling (orthonasal) or research is ongoing and might surely be regarded as one of the food uptake (retronasal). The odor quality of aldehydes varies

Figure 7. Chemical structures of b-ionone, b-damascenone, and 3-hydroxy-b-damascone-b-D-glucoside. JOURNAL OF THE AMERICAN SOCIETY OF BREWING CHEMISTS 7 strongly with carbon chain length and degree of saturation: methyl esters (e.g., methyl-4-decenoate) can be easily found in Short chain aldehydes such as (Z)-3-hexenal or hexanal are conventional hop oil and beer analysis. The introduction of characterized by intense green and grassy smells, whereas long short and medium chain acids or methyl esters into fermenting chain aldehydes such as (E)-2-nonenal or (E,E)-2,4-nonadienal wort or green beer results in a (trans)esterification into ethyl have rather papery, fatty, and deep fried aromas. Trans-4,5- esters.[15,16,18] Ethyl esters are important contributors to fruity epoxy-(E)-2-decenal, an expoxy alkenal, exhibits an intense beer flavor.[14,15,18,19] During aging of hops, the oxidation of lip- metallic odor. Short chain acids such as butanoic acid or hexa- ids and the hydrolysis of fatty acid methyl esters release short noic acid exhibit cheesy, rancid, and goat like smells, whereas and medium chain acids.[29,31,47,113] the polyenes (E,Z)-1,3,5-undecatriene and 1,3(E),5(Z),9-unde- In addition to lipid derived straight chain acids, a range catetraene are characterized by fresh balsamic odors. Structures of methyl branched short chain acids can be found in fresh of selected lipid derived odorants are shown in Figure 8.In hops, but, more importantly, in aged hops.[29] 2-Methylpro- hops, lipid degradation products receive minimal attention. panoic acid, 2-methylbutanoic acid, 3-methylbutanoic acid, This might not be due to their insignificance, but rather to the 4-methylpentanoic acid, and hexanoic acid result from the fact that those compounds occur in minimal concentrations oxidative degradation of a-acids.[47,113,114] Rettberg et al.[29] and cannot be analyzed with common analytical routes. Stein- observed a remarkable increase in the concentration of haus and Schieberle[4] identified hexanal, octanal, nonanal, (Z)- short chain acids (factor 10) during aerobic hop storage 3-hexenal, (Z)-4-heptenal, (E,Z)-2,6-nonadienal, (E,E)-2,4- and proposed a hydrolytic cleavage being responsible for nonadienal, and trans-4,5-epoxy-(E)-2-decenal in fresh and the release of these odor active compounds from odorless dried hop cones. Aroma extract dilution analysis (AEDA) has precursors. It is thought that the charge distribution of the made the remarkable odor activity of most aldehydes evident. central six-membered ring (2-acyl-1,3-diektone moiety) of In particular, the key compound of the green aroma of fresh a-acids triggers a nucleophilic attack of water onto the car- hops was attributed to (Z)-3-hexenal, whereas trans-4,5-epoxy- bonyl function group of the acyl side chain. It is supported (E)-2-decenal was identified as the most potent odorant of fresh by the findings of Rakete et al.[115] who identified a hydro- and dried hop cones (variety Spalter select) that is formed by lyticcleavageofacylsidechainsofiso-a-acids as a source linoleic acid breakdown during kilning.[4] Trans-4,5-epoxy-(E)- of monocarboxylic acids during beer aging. These acids 2-decenal has been identified in beer,[109,110] where it is accom- exhibit the intense sour, sweaty, rancid, and cheesy odors panied by sweet and malty smelling cis-4,5-epoxy-(E)-2-dece- associated with the smell of old hops. Hop derived short nal. The occurrence of cis-4,5-epoxy-(E)-2-decenal in hops has chain acids play two important roles in beer flavor.First,2- not been described so far. In general, lipid derived aldehydes methylbutanoic acid, 3-methylbutanoic acid, and hexanoic are a major “stale flavor” concern to brewers, and their occur- acid were identified as primary odorants of pilsner beer and rence is usually attributed to the breakdown of malt lipids dur- pale lager beer.[14,19] Second, they also act as precursors of – ing mashing (cf. Vanderhaegen et al.[111] for a comprehensive ethyl esters that strongly influence beer flavor.[14 16,18] Sev- review). In cases where hops are added in the brewhouse, evap- eral researchers agree that the use of aged hops rich in oration and reduction of aldehydes during fermentation is short chain acids positively affects beer flavor.[15,18] likely.[31] As for other hop derived volatiles, a minor contribu- tion of these compounds to beer flavor can be assumed. There Analytical tools of hop oil analysis and analysis of is not yet a comprehensive survey on the effect of lipid derived hoppy beer flavor aldehydes and their precursors (unsaturated fatty acids) to fla- vor (stability) of late and dry hopped beer, but Foster et al.[112] Even though the first reports on odor active hop volatiles trace showed increasing amounts of both staling aldehydes and fatty back to the 19th century, a detailed and specific analysis of hop acids in beers produced by post-fermentative dosage of (pre- essential oil and hop derived volatiles from beer started with isomerized) hop extracts. On the other hand, green aldehydic the introduction of GC–MS in the 1960s–1970s.[116] Since then, odors of fresh hops are regarded as positive flavorings. Aside numerous significant papers have been published[121,122] and from aldehydes, several methyl esters, as well as short and the evolution of powerful and sensitive instrumentation is medium chain acids, are present in hops.[29,31] Some abundant therein well documented. Today, the analysis of principal

Figure 8. Chemical structures of trans-4,5-epoxy-(E)-2-decenal, (Z)-3-hexenal, butanoic acid, (E,E)-2,4-nonadienal, hexanal, and hexanoic acid. 8 N. RETTBERG ET AL. volatiles from hop essential oil (i.e., myrcene, a-humulene, sugars. Regardless of whether volatiles from hops or beer b-caryophyllene, linalool, and geraniol) is an established tech- are analyzed, the removal of nonvolatile material using suit- nique. However, identification and quantification of minor vol- able methodologies of sample preparation prior to gas chro- atiles (e.g., thiols, aldehydes, or fatty acids) in hops, as well as matography (GC) is a prerequisite. The isolation and the analysis of hop derived volatiles in beer (e.g., for quality enrichment of volatiles for a successful analysis is critical control purposes within a brewery), remains challenging. and is, therefore, outlined in the following sections. Table 1 Although in the last 40 years of hop research, numerous excel- summarizes important features of each sample preparation lent and comprehensive papers have been published, hop technique and aims to conveniently summarize the detailed aroma in beer is still not fully understood and analytics remain information given in the following sections. Even though challenging. The analytical challenges primarily relate to the the routes applied for collecting volatiles from hops and following factors: beer may vary due to the large concentration differences in  There is no brief and universal list of compounds that is the respective mediums, the techniques of GC separation, sufficiently able to describe hop aroma and hoppy beer detection, standards, the method validation and the calibra- flavor. tion procedures have the same requirements. Consequently,  Hop aroma compounds differ in their chemical and phys- these are corporately discussed for hop and beer analysis. ical properties. Some important distinctive features are reactivity (functional groups), polarity, boiling point, and Isolation of volatiles by steam distillation molecular weight.  The concentration of hop derived volatiles in wort and Steam distillation (hydro distillation) is the most common method beer relates to hopping regime as well as overall brewing employed to isolate volatile odorants from nonvolatile (plant) technology. The concentration of individual volatiles material. In hop analysis, ground cones or pellets are boiled for sev- varies greatly, starting from sub ppb levels. eral hours; volatiles evaporate and are then either condensed and Reviewing published literature, analytical routes employed collected as an oily emulsion (total oil) or trapped in a solvent. for hop research vary greatly. Based on the availability of Diluted aliquots of the distillate are analyzed by GC with flame ion- instrumentation, multiple customized assays were published ization detection (FID) or by GC–MS. Steam distillation methods and used. All assays for hop aroma analysis from beer and hops are relatively easy to perform. With the exception of a distillation follow a basic four-step workflow; a simplified schematic out- unit and a GC–FID/MS, no costly and specialized instrumentation line is shown in Figure 9. The four principal steps in hop aroma is needed. Distillation enables an exhaustive recovery of volatiles analysis are the isolation of volatiles (sample preparation), the and the GC chromatograms of distilled hop oils are characterized gas chromatographic separation of the volatile fraction into by hundreds of signals. As well as being easy to perform, steam dis- individual compounds, the detection and identification by tillation has many advantages. The possibility of volumetric total means of physical detectors, as well as the quantification. The oil determination is highly relevant to the industry.[119] Conse- following chapters will briefly summarize the basics of these quently, steam distillation is the standard method of the American four steps, introduce the working principles of respective tech- Society of Brewing Chemists (ASBC),[120] as well as the European niques, outline their field of application (hop analysis vs. beer Brewery Convention (EBC).[121] However, steam distillation was analysis), and highlight potential strengths and weaknesses. It criticized for requiring large sample amounts, being time consum- is vital to know that some analytical tools might be used for the ing and difficult to automate, and not being capable of directly analysis of hop aroma from hops and from beer, whereas others interfacing with chromatography.[122] From a quality perspective, are only applied to one of both matrices. the formation of artifacts by oxidation,cyclization,polymerization, It is well accepted that aroma compounds in either hops hydrolysis, and isomerization of volatiles, as well the degradation or beer are embedded in complex matrices. Hops are rich of nonvolatile molecules, have been identified as the major inbittersubstances,polyphenols,lipids,[117] proteins,[118] disadvantages.[1,123,124] and carbohydrates, beer contains relevant concentrations of In order to gently recover hop oil and shorten the time for fermentation by-products, nonvolatile bitter acids, and sample preparation, several alternative methods have been

(a) Isolation (b) Separation (d) Quantification

(c) Detection + Identification

Figure 9. Simplified schematic workflow of an assay for the analysis of aroma compounds. The principal steps include (a) the isolation of volatiles from bulk matrix, (b) the separation of a complex mixture into individual compounds, (c) their detection and identification by means of physical detectors, as well as (d) the quantification. JOURNAL OF THE AMERICAN SOCIETY OF BREWING CHEMISTS 9

Table 1. Tabular compendium of sample preparation techniques frequently used in hop and beer analysis.a

Environmental Field of application Practical aspects aspects Publications

Hop Beer Direct GC Sample Need for Selected analysis analysis interfacing Automation volume organic references solvents

Steam distillation X No No 10 g No 3, 22 Simultaneous distillation extraction (SDE) X X No No 10 g / 300 mL Yes e.g. 13, 81, 92, 121, 154 Liquid–liquid extraction (LLE) X X No No 5 g / 300 mL Yes e.g., 33, 48, 66, 75, 77, 162, 163, Liquid–solid extraction (LSE) 166 Solid phase extraction (SPE) Direct thermal Desorption (DTD) X Yes Yes < 1 g No 21 Static headspace (HS) X X Yes Yes < 1 g / 20 mL No 26, 31, 56 Headspace solid phase microextraction X X Yes Yes < 1 g / 20 mL No e.g., 26, 40, 70, 96, 112, 114, 140 (HS-SPME) Dynamic headspace (DHS) X X Yes Yes < 1 g / 20 mL No e.g., 1, 6, 129, 131 Headspace trap (HS-trap) Stir bar sorptive extraction (SBSE) X Partly Possible 100 mL No e.g., 5, 64, 65 a The estimation of practical and environmental classification is based on information provided by publications covered within this review. Selected references are pre- sented to simplify familiarization with details of the respective techniques that are beyond the scope of this paper. evaluated. These include vacuum steam distillation[125] and steps of sample preparation and can be easily automated. Salt supercritical carbon dioxide extraction,[126] as well as the addition, shaking or stirring, as well as heating are usually extraction of hops using solvents of various polarities.[66] applied to shift equilibrium and to speed up gas phase satura- Whereas solvent extraction can only be poorly automated, the tion. HS-GC is an equilibrium extraction and its major disad- major disadvantage of vacuum steam distillation or supercriti- vantage is the limited sensitivity. Volatiles occurring in low cal carbon dioxide extraction is the need for rather costly, spe- concentrations or substances with high boiling points (or in the cialized equipment. Generally, methods of nondistillation are worst case a combination of both) are not sufficiently either prone to incomplete recovery of oxygenated water solu- enriched.[130,131] In hop analysis, the lack of sensitivity limits ble compounds or to carryover of nonvolatile compounds such the application of HS-GC to the analysis of abundant as bitter acids.[127] Whereas the problems of incomplete recov- volatiles.[132,133] ery can be reduced by application of suitable internal standards, In order to overcome the limited sensitivity of static HS-GC, residues of nonvolatile material rapidly reduce the performance but to take advantage of its strength to gently separate volatiles of GC columns and stress sensitive parts of the mass spectrom- from nonvolatiles, headspace solid-phase microextraction (HS- etry (MS; e.g., ion source). Consequently, these methods are SPME) was introduced. In HS-SPME, a rod shaped adsorbent, not very popular for sample cleanup prior to routine analysis, called fiber, is exposed to the headspace above a sample. These but are suitable for special preparative purposes.[128,129] reusable fibers, made from (mixtures) of polydimethylsiloxane, divinylbenzene, and CarboxenÒ, enrich volatiles and increase sensitivity by a factor of 10 to 20 compared to HS-GC. In accor- Automated sample preparation techniques for hop aroma dance with HS sampling, HS–SPME is an equilibrium extraction analysis technique; partition of analytes between matrix, headspace, and Automation is the buzzword of analytical chemistry. It sug- fiber determines the extraction efficiency for each individual gests that a reduction of manual handling reduces costs and analyte. The amount of analyte enriched on the fiber, namely improves analytical performance by increasing reproducibil- the mass transfer (n), is directly related to the analyte concentra- fi ity, accuracy, and stability. Automation sees remarkable tion in the sample (C0), the sample (Vs) and ber (Vf) volume, reductions of sample volume, while sophisticated and selec- as well as the partition coefficient of the analyte among the fi tive enrichment techniques are applied in order to maintain matrix, headspace, and ber coating (Kfs) (cf. formula 1). sufficient sensitivity. Several automated and miniaturized sample preparation techniques are available for hop and D KfsVf C0Vs n C beer analysis. The applicability of each method basically KfsVf Vs dependsontargetconcentrationandonthenatureofthe sample (beer/liquid or hops/solid). In SPME, sample matrix and fiber compete for analytes, In the analysis of volatiles, the most gentle and straightfor- thus the choice of appropriate fiber material is the funda- ward isolation technique is any form of headspace (HS) analy- mental step in manipulating extraction efficiency toward sis. HS-GC is based on the thermodynamic equilibrium certain analytes (high Kfs). The extraction speed, an impor- between the mixture of volatiles in the headspace above the tant factor of routine analyses, is controlled by mass trans- sample and in the sample itself in a closed system (i.e., HS-GC- fer from the sample into the headspace, followed by vial). As soon as equilibrium is reached, usually <30 min, an adsorption onto the coating and then absorption and diffu- aliquot of the headspace is injected into GC where the volatiles sion into the coating. In liquid samples, salt addition, stir- are separated and detected. HS-GC does not require additional ring, and heating can improve the mass transfer into the 10 N. RETTBERG ET AL. headspace. These techniques are less effective in solid sam- to 400 mg/L. Beer contains multiple, abundant fermentation ples such as hops. In fact, mass transfer from solid samples by-products (mg/L range) with chemical properties (e.g., boil- into headspace is slow, which strongly limits HS and HS– ing point and polarity) identical or at least very similar to hop SPME performance. Although it is way more suitable for derived odorants. In addition, the structural diversity of hop the analysis of volatiles in beer, HS–SPME is often also derived aroma compounds and the reactivity of some targets used in hop (oil) analysis because of its simplicity of sam- add remarkable complexity to isolation, separation, identifica- – pling and straightforward automation.[134 137] tion, and quantification. In addition to HS–SPME, which for a long time was exclu- The first occurrence of hop derived odorants in beer was sively patented to SupelcoÒ, instrument manufactures devel- published by Likens and Nickerson in 1964.[142] They oped multiple sampling techniques aiming to increase the enriched volatiles from beer by so-called simultaneous dis- enrichment of volatiles from headspace systems. These techni- tillation extraction (SDE), a method today widely known as ques are called dynamic headspace (DHS) or headspace trap the “Likens and Nickerson method.” The SDE uses a special (HS-trap) techniques. Here the enrichment of volatiles is distillation unit, in which steam distillation and extraction increased by purging the sample headspace with a controlled of volatiles into a solvent occurs simultaneously.[142,143] flow of inert gas. The purged volatiles are then trapped on a After its publication in 1964,[142] the original SDE setup replaceable adsorbent-filled trap, which is thermally desorbed was modified and miniaturized. In either way, it has been to release volatiles into the GC. In order to efficiently enrich successfully used to evaluate volatile fingerprints of various volatiles, multiple purge cycles may be performed to load the foods, drinks, herbs, and fruits (see Chaintreau[144] for a trap prior to desorption, and, by this, a concentration in order comprehensive review). Targeting the analysis of hop to effectively transfer the total vapor of a sample to the GC col- derived compounds in beer, the major advantage of SDE is umn. Even though dynamic headspace sampling was initially its rather low specificity and the possibility to execute an developed for the analysis of liquid samples,[138,139] these meth- exhaustive isolation of the volatile fraction. In SDE enrich- ods have been successfully applied for hop analysis. This can be ment of volatiles relies on their boiling points and steam carried out either after extraction with an organic solvent[1] or volatility, the selection of solvents varying in polarity can by direct analysis of ground samples.[140] impact the recovery of specific substance groups. Here, Another alternative method to HS, HS–SPME, and DHS is dichloromethane, diethyl ether, and low boiling point alka- direct thermal desorption (DTD). DTD requires minimal sam- nes (e.g., pentane) are most suitable. SDE yields an aroma ple preparation, namely grinding of inhomogeneous samples. extract that might either be directly analyzed or further In DTD, the isolation and concentration of volatiles from a concentrated, also further processing by chromatography or nonvolatile matrix relies on a two-step procedure: Initially, vol- liquid–liquid extraction (LLE) is useful when analytes of atiles are purged from the sample, and then they are trapped by low concentration are focused.[31] GC can be performed means of adsorbent materials by cryo-focusing. Even though using standard spilt-splitless injection units. SDE aroma DTD and DHS appear to be similar, the basic difference is that extracts are stable if stored cold (e.g., at ¡20C), which ena- in DTD the sample material itself, and not the headspace above bles uncoupling of sample preparation and analysis. As a sample, is purged. Practically, a DTD protocol can be carried accounts for steam distillation of hops, the major technolog- out as follows: 1 g ground hops is placed into either glass or ical drawback of SDE is the thermal impact on the sample. steel desorption tubes. These are then purged by a flow of inert Well-known examples of heat artifacts are the decomposi- carrier gas (e.g., helium at 150C for 5 min).[141] In order to tion caryophyllene oxide[1] or the release of aglycones from increase analyte recovery, purging can be intensified by longer nonvolatile material.[84] Surely, the thermal load can be purging times. The theory of DTD is simple, compared to DHS reduced when shortening extraction time, but from an eco- enrichment of volatiles is faster, since no equilibration between nomic (manual handling) and environmental (solvents) headspace and sample is required. From a quality perspective, viewpoint SDE seems unfashionable. both DHS and DTD show good recovery of reactive and labile An alternative to eliminate thermal load is the extraction by volatiles, as well as substances with high boiling points. Results LLE or LSE. Methods to extract aroma compounds from beer from hop analysis using DTD and DHS correlate with results use either solid-phase extraction (SPE),[72] LLE,[4,5,14,19] or from steam distillation at first view. However, data for the indi- LSE.[17,34,50] Even though published extraction protocols are vidual targets differs considerably. Detailed comparative studies rather simple to implement, one should neither overestimate are given by Aberl and Coelhan.[1] and Eri et al. [141] performance nor underestimate time and effort. Technically, the major problem of SPE, LLE, and LSE remains the effective enrichment of oxygenated and water soluble molecules. In order Challenges in isolation of hop derived volatiles from beer to extract those, relatively high amounts of (harmful) solvents as The analysis of hop derived aroma compounds in beer is even well as numerous repeat extractions are required. Depending on more challenging than the analysis of their analogues from the organic solvent used for extraction, phase separation (beer- hops. Brewing leads to a considerable dilution of hop derived solvent) not only requires centrifugation, but also special techni- volatiles; chemical and biochemical modifications of hop aroma ques such as solvent assisted flavor evaporation (SAFE), distilla- compounds yield multiple analytical targets. Whereas in pilsner tion or Kunderna-Danish evaporation.[14,19,145] Whereas type beers and pale lager beers, target concentrations are in the dichlormethane is frequently used prior to GC–MS analysis,[9] range of 1 to 10 mg/L, in dry hopped beers some analytes (e.g, diethyl ether followed by the SAFE distillation is also suitable for linalool and geraniol) can accumulate to concentrations of up the sample cleanup prior to GC–Olfactometry analysis. JOURNAL OF THE AMERICAN SOCIETY OF BREWING CHEMISTS 11

In order to eliminate manual handling and solvent use, but with limited fiber volume (typically less than 0.5 mL adsorbent) most importantly to implement powerful automated assays for challenge the analyst. These HS–SPME drawbacks can be circum- beer analysis, three important extraction techniques are practi- vented by use of the so-called SBSE. SBSE relies on the selective cally applied. In accordance to hop analysis DHS/HS-traps and adsorbance and absorbance of volatiles onto/into a stir-bar, which HS–SPME are frequently used, additionally stir bar sorptive in most cases is coated with Polydimethylsiloxan (PDMS) that is extraction (SBSE) is used. stirred in liquid samples.[15,33,151] The theory of SBSE is similar to In beer analysis, HS–SPME emerged as the most frequently that of HS–SPME and the extraction efficiency for each analyte [6,7,10,12,128] – used method for sample preparation. HS SPME in depends on the Kfs of the analyte between matrix and stir bar. – combination with GC MS is sensitive enough to cover most of When using stir bars coated with PDMS, Kfs is proportional to the the relevant hop derived analytes and enables additional rele- octanol-water coefficient. The major advantages of SBSE are the vant applications such as carbonyl analysis from beer.[146] phase ratio between sample and stir bar coating and the direct con- Although its flexibility makes HS–SPME a powerful tool in tact between stir bar and sample. In SBSE, up to 100 mLofPDMS beer analysis, several crucial factors need to be considered are applied for target enrichment, thus an increase in sensitivity when quantitative analysis of aroma compounds from complex compared with HS–SPME can be observed.[33,151] SBSE is an effi- matrices is performed.[131] On the one hand, HS–SPME is sen- cient tool for beer analysis, but the applicability to solid samples is sitive to any changes in experimental conditions. The target limited. In process and quality control, flexible instrumentation is enrichment depends strongly on assessable extraction condi- required to cope with samples of the whole beer production chain. tions such as temperature, exposure time, fiber material, fiber Consequently, an instrumentation that enables the analysis of capacity, agitation, sample, and headspace volume, but most odorants from both solid and liquid samples (preferably in a row importantly on uncontrollable sample characteristics. Samples without the need for instrumental changes) is always preferred. might strongly vary in ethanol content (from wort without This flexibility is a major advantage of HS-trap technology as previ- alcohol to India pale ale with >6% ABV) or in the concentra- ously described. HS-trap has been successfully used in hop aroma tion of volatiles (e.g., fermentation by-products or hop aroma analysis,[1,140] and, most recently, the analysis of hop-derived compounds). While the concentration of ethanol influences the aroma components in beer was reported by Schmidt and concentration of minor odorants in sample headspace, high Biendl.[152,153] Although the HS-trap technique has not been widely concentrations of volatiles can cause competitive effects in used thus far in beer analysis, this technology has the huge advan- respect to adsorption onto fiber. On the other hand, HS–SPME tage of trapping hop derived volatiles from cone to the glass. is a selective extraction technique. That means the target enrichment strongly depends on the physical-chemical proper- Separation by GC ties of the analyte, namely volatility, water solubility, and molecular weight, as well as the Kfs of the analyte between Aroma active volatiles from hops and beer are separated by GC. matrix, headspace, and fiber. It has to be emphasized that mini- Since relevant analytes are spread over a wide polarity range, a mal variations in the chemical structure of analytes (down to wide range of stationary phases are suitable and practically the stereochemistry) can lead to remarkable changes in their used. The most common phases are basic polar (polyethylene HS–SPME recovery and might result in misinterpretations in glycol) and nonpolar capillary columns (polyethylene glycol improperly calibrated systems. A simple example might illus- including 5%-phenyl-methylpolysiloxane), but also enantiose- trate this: The recovery of geraniol (BP D 229C) from model lective phases have been successfully applied in the analysis of wine was found to be higher by a factor of 1.6 than its isomer chiral compounds in hop oils and hopped beers.[4,102,154] Gen- linalool (BP D 198C) and lower by a factor of 0.82 compared eral declarations on the performance of one or the other GC- to nerol (BP D 225C) using a polyacrylate fiber. Comparison phase are hard to give, but in the analysis of beer samples con- of ethyl hexanoate (BP D 168C) and ethyl octanote (BP D taining multiple, abundant, and polar fermentation by-prod- 172C) resulted in a difference in recovery of a factor almost ucts (e.g., organic acids), the polar phases might be favored. It 10 times higher for ethyl hexanoate.[147] Those remarkable is noncontroversial that co-elution of some analytes cannot be differences indicate the multiplicity of parameters influencing avoided and that symmetry of some signals is poor. Multi-ana- matrix-headspace-fiber-analyte interactions and, consequently, lyte methods using one-dimensional GC represent a reasonable the transfer from sample into the chromatographic system. In compromise, rather than a perfect solution. Indeed, certain co- addition to matrix effects caused by abundant volatile and non- elution problems might be minimized by MS detection. How- volatile beer constituents, a decrease of sorptive capacity of the ever, if substances with similar chemical structure and MS fiber (fiber aging) should be considered.[148,149] In brief, qualita- properties co-elute, then there is false identification and misin- tive HS–SPME analysis is rather straightforward, whereas terpretation of mass spectral data, and, consequently, false quantitative HS–SPME requires reliable internal standards quantification is likely. When GC is coupled to olfactometric D – (Kfs analyte Kfs standard). The general consensus is that in detection (GC O), additive effects of co-eluting volatiles might HS–SPME, stable isotope labeled standards are the superior lead to incorrect estimation of odor activity and quality. Hence, choice to fully exploit the potential of this extraction for certain research purposes, two-dimensional gas chromatog- à à technique.[131,150] raphy (GC GC) might be applied. In GC GC, two different sta- The major drawback of HS–SPME is the nonquantitative tionary phases are connected by a cryogenic modulator. extraction. Practically, semi-volatile and water soluble compounds Sequential trapping and pulsing of effluents from the first to or targets those with high boiling point analytes are hardly recov- the second column result in narrow peaks and a two-dimen- ered. Limited mass transfer from matrix into headspace combined sional separation. Practically, two compounds with similar 12 N. RETTBERG ET AL. boiling points that co-elute on the first column can be resolved determination of unknown substances in GC requires two à on the second column if they differ in their polarity. GC GC independent forms of identification: In GC–FID analysis, results in greater peak capacity, higher resolution, and sensitiv- retention times of analyte and standard on two different ity. Thus, this technique can provide superior analyses com- chromatographic columns (polar C nonpolar) are used, à pared to single-column GC. GC GC has been successfully whereas in GC–MS the retention time and mass spectral applied to the analysis of hop oils and beer samples; however, need to match.[158] Since retention times vary depending due to the complexity of those analytical systems, its applica- on the temperature programming of the GC, and they are tion in a high throughput environment, such as routine quality sensitive to instrumental changes (e.g., column replace- – control in a brewery, does not seem practical.[9,131,155 157] ment), relative retention indices are widely used.[159] Although substance identification using modern GC–MS equipment is convenient, problems caused by operator Detection and identification of aroma compounds by reliance on structures predicted by mass spectral libraries means of physical detectors frequently occur. There are over 230 sesquiterpenes with a FID is the most common detector coupled with GC. In molecular mass of 204 in nature and most exhibit similar cases where analytical targets are well characterized and fragment ions for MS identification. Therefore, any identi- occur in high quantities, and where GC separation perfor- fication needs to be supported with at least retention indi- mance is evidently good, substance identification can be ces, whereas the analysis of authentic reference materials carried out by matching the retention times of analytes under analysis conditions is still the superior tech- with reference materials and calculation of relative retention nique.[160] For the sake of completeness, it has to be indices. For distilled hop essential oil, in which the concen- remarked that the detection of sulfur containing com- tration of the main targets is high, GC–FID is a simple and pounds is still a challenge. The detection is usually carried robust technique that enables relative quantification. When out using MS instrumentation, flame photometric detectors beer samples are analyzed, the GC–FID performance is (FPD), pulsed flame photometric detectors (PFPD), sulfur rather limited. It requires an effective and selective enrich- chemiluminescence detectors (SCD), or atomic emission ment technique (such as HS–SPME) in order to concentrate detectors (AED). the target compounds and remove the matrix. When it comes to the analysis of minor volatiles from hops or trace GC–O concentrated volatiles from beer, GC–MS is undoubtedly the method of choice. GC–MS enables the acquisition of The majority of aroma related research of raw materials mass spectral data that permits substance identification and (hops) or final products (beer) is based on the identification is also able to detect signal overlaps (co-elution). Depending and quantification of volatiles by means of physical on the type of the mass spectrometer, mass accuracy, reso- detectors (FID or MS) in combination with sensory analy- lution, and detection modes differ. Whereas accurate mass sis. In the interpretation of these approaches, one aims to determination by time of flight (TOF) detectors is a power- correlate the increase or decrease of one or the other aroma fultoolintheidentification of (new) metabolites, the cost compound to changes of the aroma profile of a product.[9] effective quadrupole mass spectrometers are widely used for Since odor perception (e.g., thresholds) varies widely among routine applications. Quadrupole mass spectrometers (QP– volatiles, the concentration of a volatile and its relevance MS) are the most common mass selective detectors coupled (aroma contribution) do not necessarily correspond. The to GC, and they are either operated in so-called “scan” or GC–O is used in order to identify odorants qualitatively “selected ion monitoring” (SIM) mode. In scan mode, all and to establish measures to quantify their odor activity. In ions of a defined range (e.g., m/z D 29 to 400) are detected GC–O, human assessors detect and evaluate volatile com- and several complete mass spectra are recorded per second pounds as they elute from the GC.[155] A detailed discussion of data acquisition. Whereas scan mode (in combination of GC–O techniques is beyond the scope of the paper; refer with either electron impact ionization (EI) and chemical to Plutowska et al.[161] for a review. Nevertheless, it must be ionization (CI)) are used for untargeted substance identifi- underlined herein that GC–O, preferably in combination cation, SIM–MS is exclusively used for targeted identifica- with GC-MS, is an extremely powerful technique of hop tion and quantification. In SIM–MS, a limited number of research. Combinations of GC–MS and GC–Oareusedin characteristic and abundant ions for each target compound both aroma extract dilution analysis (AEDA)[162,163] and are selected; by reducing the number of detected ions the Charm analysis[14,19] for hop and beer aroma.[4,5,16,17] sensitivity of detection can effectively be increased. How- ever, special attention must be paid to method development, Quantification procedures use, and calibration, since the prerequisite of SIM–MS is the availability of authentic reference materials. In SIM–MS, it In analytical chemistry, quantitative analysis stands for the is vital to frequently compare ion ratios of target com- determinationofabsoluteorrelativeabundanceofoneor pounds with those of reference standards, otherwise identi- multiple substance(s) presentinasample.Inmostcasesthe fication and quantification fails. When any MS detection is abundance is expressed as a concentration in %, mg/kg, mg/ available, stable isotope labeled standards might be used kg, mg/L, or mg/L. In chromatography, any quantification is for quantification. A brief summary of the advantages of based on the response of physical detectors, named peaks. this technique is provided as follows. Generally, In order to relate the peak intensity (area) in a JOURNAL OF THE AMERICAN SOCIETY OF BREWING CHEMISTS 13

Figure 10. Comparison on the quantification procedures employed in the analysis of volatiles (here linalool) from hops and beer. chromatogram to the concentration of a substance in the The choice of a quantification procedure relates to multi- sample, several workflows might be used. A brief descrip- ple factors. A consideration of expected substance concen- tion of the basic setup of different quantification proce- tration (working range), the instrumental setup, and the dures, as well as a discussion of their strengths or protocol of sample preparation, as well as the matrix to be weaknesses is outlined in the following sections. Figure 10 analyzed, are of primary importance. As the quantification represents a schematic illustration of different calibration is based on detector response, it is important to consider protocols. their working principles. The response of physical detectors 14 N. RETTBERG ET AL. such as FID and MS not only depends on substance con- other matrix constituents. They rely on calibration protocols centration, but also on the chemical structure and the ele- that create a relation between detector signal (peak area) and mentary composition of the analyte. The principle of FID is substance concentration. Any calibration relies on the analysis simple: Compounds eluting from the GC are combusted by of reference standards of known concentrations. There are dif- ahydrogenflame and ions are formed. Those ions are col- ferent calibration protocols, which primarily differ in the type lected on an electrode and produce an electrical signal that of reference standard used and conditions under which the cali- is proportional to the concentration of organic species gas bration is recorded. in the gas stream. The response of FID shows slight varia- tions related to functional groups present in the molecules. External calibration Setting the response of hydrocarbons (e.g., octane) to 1.0., In analytical methods requiring either no or only minimal sam- the detector response is 1.3 for alcohols and 1.4 for alde- ple preparation, external calibration might be performed. hydes. This response remains constant over a wide linear External calibration means, that several dilutions of reference range (107), which facilitates quantification using FID. In standards (mostly five) are analyzed in separate runs and cali- mass spectrometry, response factors vary over a greater bration graphs (plot of relative area counts against substance range, and linearity is rather limited.[164,165] This relates to concentrations) are established. For the calibration parameters construction and working principle of these instruments of sample preparation (i.e., extraction time, sample, or head- whose description exceeds the scope of this review. In prac- space volume) and GC are adjusted to match those applied dur- tice, MS instrumentation is sensitive and changes in the ing sample analysis. In order to consider variations in absolute and relative ion counts can be observed over time. instrument performance (e.g., a decreasing fiber capacity in For example, this relates to fouling of ion source and HS–SPME), frequent calibration is required. requires frequent checks of MS performance. The control of External calibration is rather simple to perform; the only mass accuracy and ion source tuning are basic measures to basic requirement is the availability of pure reference standards. obtain constant analytical conditions. A detailed survey of The downside is that both accuracy and consistency are ques- those factors has been written by Pollnitz et al.[166] Refer to tionable when one aims to quantify volatiles from complex Niessen,[167] McEwen et al.,[168] and Grob and Barry[169] for matrices. Different hop products or different types and styles of textbooks on the current practice of GC and GC–MS beer strongly vary in their chemical composition. Depending on operation. the type and selectivity of sample preparation, the recovery of one or the other analyte may be disproportionate. These so- called matrix effects can influence the isolation of minor volatiles Relative quantification and is neither considered nor equalized by basic external calibra- In the analysis of hop essential oil the ASBC and EBC standard tion.[131] In order to improve its significance, external calibration methods rely on a so-called “relative quantification.”[120,121] In should be carried out in a matrix as similar as possible to the these methods, an aliquot of hop oil is analyzed by GC–FID, and sample. This procedure is called matrix matched calibration. By peak areas are then used to calculate the relative percentile con- applying matrix matched calibration, one basically determines tribution of single aroma compounds to the share of total vola- the recovery of analytes in a given system. This includes a con- tiles. If the concentration of total volatiles (hop oil) in a sample sideration of extraction efficiency and detector response. is known, the concentration of individual aroma compounds in the original sample (e.g., hop pellets or cones) can be calculated. Internal calibration This procedure of relative quantification is simple and reli- As described previously, the comparison between analyte signal able as long as: (peak area) and standard signal (peak area) is only meaningful  one aims to quantitate the major volatiles of hops or hop oil if both were created under matching experimental conditions. (e.g., myrcene, b-caryophyllene, a-humulene, and linalool) This is hardly the case in external calibration, where variations  an exhaustive sample preparation (e.g., steam distillation) in matrix are generally not considered. is employed Hence, it is accepted that many calibration problems can be  samples injected into GC exclusively contain volatiles reduced by using internal standards.[170] In internal standard (e.g., essential oil) assays, a substance similar to the analyte, but not originally con-  slight variations in detector response (i.e., due to the tained in the sample, is added to the sample prior to the sample occurrence of functional groups) can be neglected. preparation. Along with the compounds of interest (e.g., terpe- Considering these factors, a relative quantification is impre- noids in beer), the internal standard undergoes the entire analytical cise or not applicable when minor constituents of a sample process. By this, any variations in sample preparation and analysis shall be analyzed, equilibrium extraction techniques are are indicated, whereas the internal standard is the scale basis. applied, or mass spectrometric detection is used. In those cases, The quality of internal standard rises with structural similar- which apply to any hop aroma analysis in beer, protocols that ity to the analyte (matching chemical, physical, and mass spec- enable a “true” quantification of volatiles are required. trometric properties), and if the used standard concentration is similar to the expected analyte concentration.[164] Conse- quently, methods that aim to quantify multiple odorants might “True” quantification need several internal standards. “True” quantification procedures aim to report concentrations The workflow to calibrate an internal standard assay is basi- of selected aroma compounds, disregarding the presence of cally in accordance to the workflow described for the external JOURNAL OF THE AMERICAN SOCIETY OF BREWING CHEMISTS 15 calibration. The fundamental difference is that diluted refer- technique has been written by Garbe and Rettberg[127] Briefly, ence standards are spiked with the internal standard of con- the advantages of SIDA relate to the identical physical and stant concentration. As accounts for external calibration, chemical properties of analyte and stable isotope labeled stan- internal calibration is preferably carried out in the matrix of dard. This assures equal behavior during sample cleanup (e.g., – interest (e.g., wort or beer). Examples for internal standards matching recovery rates or Kfs in HS SPME), and chromatog- used in hop aroma analysis from beer are naphthalene,[28,46,63] raphy. In MS, the fragmentation intensities of analyte and iso- cis-hepten-1-ol,[33] and dodecane.[94] tope standard are similar and quantification can be done by direct comparison of the respective m/z. Figure 11 shows the Stable Isotope Dilution Analysis (SIDA) electron impact (EI) mass spectra of linalool and a D5-labelled The accuracy and robustness of an internal standard assay rises stable isotope standard. The mass to charge ratios (m/z) of with the structural similarity of standard and analyte. Conse- abundant and characteristic ions (only) differ due to the intro- quently, the most reliable standard for quantification using duction of deuterium labels. Identification and quantification GC–MS is a stable isotope labeled analogue (isotopologue) of of analyte and standard can be performed by choosing similar the target compound.[131] This technique is called stable isotope quantifier and qualifier ions. The use of stable isotope labeled dilution assay (SIDA) and is widely used in food chemistry, standards is accepted as the superior technique for quantitative food safety, drug, and environmental analysis. In those applica- analysis as systematic errors that arise from external and inter- tions, the analytical targets are usually in low concentrations nal calibration are minimized. However, no analysis is fool- and embedded in complex matrices, factors that also apply for proof, and even SIDA might yield precise but inaccurate data the analysis of (minor) volatiles from hops and beer. Several in the case of artefacts that are formed by inappropriate sample SIDAs have been published involving the analysis of volatiles preparation or harsh analysis conditions.[166] from hops and beer.[1,14,19,36,92,109,110,131,154,171,172] Stable isotope standards are not only used for quantification in food and Summary brewing chemistry, but also to elucidate chemical and biochem- ical reaction pathways. A review on the most relevant papers Hop essential oil is a complex mixture of several hundred vol- employing SIDA for the analysis of beer and raw materials, as atiles. It not only includes abundant terpene hydrocarbons well as a basic introduction into the fundamentals of this and flavor active terpenoids, but also monocarboxylic acids,

[%]

100 71 43

80

93

60

40

20 121 154a

136

0 30 40 50 60 70 80 90 100 110 120 130 140 150 m/z

[%]

74 100

80 46

60 98 85

40

20 159a 126

0 30 40 50 60 70 80 90 100 110 120 130 140 150 m/z

a Figure 11. Electron impact mass spectra of linalool (top) and D5 linalool (bottom). Labeled ions show a mass shift indicating the presence of deuterium labels. Signals of the molecular ions at m/z D 154 (linalool) and m/z D 159 (D5 linalool) are not visible due to image size. Their intensity is <2%. 16 N. RETTBERG ET AL. methyl esters, sulfur compounds, and norisoprenoids. During Dilution Techniques. J. Agr. Food Chem. 2000, 48, 1776–1783. ripening, processing, and storage of hops, as well as in the DOI:10.1021/jf990514l. brewhouse, some volatiles are evaporated and oxidized. Dur- [5] Steinhaus, M.; Wilhelm, W.; Schieberle, P. Comparison of the Most Odour-active Volatiles in Different Hop Varieties by Application of ing beer fermentation and maturation yeast catalysis and low a Comparative Aroma Extract Dilution Analysis. Eur. Food Res. beer pH trigger rearrangement, hydrolysis, reduction, or Technol. 2007, 226,45–55. DOI:10.1007/s00217-006-0507-6. esterification reactions. A large share of hydrophobic com- [6] Dresel,M.;VanOpstaele,F.;Praet,T.;Jaskula-Goiris,B.;VanHolle,A.; pounds is lost by stripping and adsorption onto the biomass. Naudts,D.;DeKeukeleire,D.;DeCooman, L.; Aerts, G. Investigation of Consequently, quality and quantity of hop addition, as well as the Impact of the Hop Variety and the Hopping Technology on the Analytical Volatile Profile of Single-hopped Worts and Beers. technical and technological variables in a brewing process, BrewingSci.–Monatsschr. Brauwiss. 2013, 66, 162–175. generate a specificvolatilefingerprint. The aroma of hops and [7] Forster, A.; Gahr, A. On the Fate of Certain Hop Substances during the flavor of hoppy beers cannot be measured by analysis of a Dry Hopping. BrewingSci.–Monatsschr. Brauwiss. 2013, 66,93–103. few key components. 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