Comparison of Hops’ Aroma Profiles Using TD–GC–TOF MS with Select-eV Variable-Energy Electron Ionization Technology

Many types of organic ingredients are found in beer, with concentrations ranging many orders of magnitude. A variety of plants contain essential oils that include aroma-active hydrocarbons such as sesquiterpenes (C15) and monoterpenes (C10).

Interestingly, these hydrocarbons are present in hops that render the typical bitterness to the finished beer. Monoterpene β-myrcene plays the most major role in beer, along with the α-humulene, β-farnesene, and sesquiterpenes caryophyllene. Besides these compounds, countless number of other terpenes may be present which could affect the final flavor and aroma.

As these compounds exhibit extremely low odor thresholds, a highly sensitive analytical method is required to evaluate the hops’ quality before initiating the brewing process. Hops’ VOC profiles can be affected by several factors, such as ageing, packaging, storage, and seasonal differences. As a result, robust quality control should be applied.

This article shows how the high-performance capabilities of a TD–GC–TOF MS system help in studying complicated aroma profiles from hops. Here, Markes’ Micro-Chamber/ Thermal Extractor™ (µ-CTE™) is used for dynamic headspace sampling of hop ‘cones’ and this followed by thermal desorption (TD) analysis.

Thermal desorption provides pre-concentration of the aroma compounds. By linking to time-of-flight MS detection with Select-eV® variable-energy ionization technology, a complete aroma profile can be studied in one sequence.

Background to BenchTOF Systems

Markes’ BenchTOF™ time-of-flight mass spectrometers have been particularly designed for gas chromatography (GC). These instruments allow fast, robust, and trace-level detection of compounds in hops for the following reasons:

  • Sensitivity: Using the advanced direct-extraction technology, the BenchTOF systems are capable of achieving full-range spectra with sensitivity similar to SIM, and thus can identify unknowns and trace-level targets in just one run, which would not be possible or may prove difficult on a quadrupole instrument.

  • Speed: The BenchTOF system is capable of recording full-range mass spectral data to very high densities, which are as high as 10,000 transient spectral accumulations every second. This allows data-mining algorithms and sophisticated spectral deconvolution to obtain maximum amount of data from signals that are weak and masked by matrix.

  • Spectral quality: The BenchTOF creates reference-quality spectra that match closely with those found in commercial libraries, for instance Wiley or NIST. This feature allows for fast and assured matching of analytes.

Experimental Framework

An overview of the analytical workflow is shown in Figure 1.

Analytical procedure used for the characterization of hop aroma.

Figure 1. Analytical procedure used for the characterization of hop aroma.

Sampling

With the aid of Markes’ µ-CTE, dynamic headspace sampling is carried out for three varieties of hops, namely Target, Goldings, and Fuggle.

About 1g of hops was placed in pots and these were controlled by temperature and separately sealed within the µ-CTE. Then, using a dynamic headspace process, volatiles were acquired at 30°C temperature for about 30min, and the same were collected onto an inert-coated stainless steel sorbent tube filled with Tenax® TA.

TD–GC–TOF MS technique was then used to analyze the tubes under the following conditions:

TD:

  • Instrument: TD-100™ from Markes International
  • Pre-purge: 1min at 20mL/min to split
  • Dry purge: 1min at 50mL/min
  • Tube desorb: 10min at 280°C and 50mL/min trap flow (no split)
  • Pre-trap fire purge: 1min at 50mL/min
  • Focusing trap: Tenax TA
  • Trap high: 280°C
  • Trap low: 25°C
  • Trap heating rate: Maximum hold for 5min
  • Split flow: 150mL/min, collected onto a clean Tenax TA sorbent tube

GC:

  • Column: HP-5ms™, 30m × 0.25mm × 0.25µm
  • Carrier gas: Helium, 1.2mL/min
  • Total run time: 70min
  • Oven temperature: 40°C for 5min, then 4°C/min to 280°C (hold for 5min)

TOF MS:

  • Instrument: BenchTOF-Select™ (Markes International)
  • Mass range: m/z 35–500
  • Data rate: 5Hz
  • Ion source: 230°C Transfer line: 280°C Filament voltage: 1.8 V

Software:

For data processing and instrument control, Markes’ comprehensive TOF-DS™ software package was utilized.

Results and Discussion

Using µ-CTE–TD–GC–TOF MS, the aroma profiles for three varieties of hops were taken (Figure 2). As predicted, all three samples contained mostly caryophyllene, β-myrcene, and α-humulene; however, β-farnesene was found only in the Fuggle variety, indicating that it may have an increased level of floral aroma.

TD–GC–TOF MS (TIC) chromatograms for each hop variety, and pie-charts showing the relative abundances of five key aroma compounds.

Figure 2. TD–GC–TOF MS (TIC) chromatograms for each hop variety, and pie-charts showing the relative abundances of five key aroma compounds.

During analysis, near-real-time data-processing was performed, thanks to the comprehensive TOF-DS™ software suite. While the specimens were still acquiring, the chromatograms were background-subtracted, incorporated, deconvolved, and finally library-searched.

This helped in reducing the time spent on data review. As acquisition went on, the entire samples were screened against the NIST 14 library, and comparison was made on the resulting peak tables. A match factor of greater than 750 was exhibited by all identifications. These identifications can be selected through chromatogram expansions (Figure 3).

Expanded views of the TD–GC–TOF MS chromatograms comparing the aroma profiles of three varieties of hops, with key compounds shown. hops, with key compounds shown.

Expanded views of the TD–GC–TOF MS chromatograms comparing the aroma profiles of three varieties of hops, with key compounds shown. hops, with key compounds shown.

Figure 3. Expanded views of the TD–GC–TOF MS chromatograms comparing the aroma profiles of three varieties of hops, with key compounds shown. hops, with key compounds shown.

It was evident that the Goldings and Fuggle varieties shared similar content, whilst the Target variety had a substantial difference in content. Conversely, all three varieties were found to vary with respect to the availability of these compounds, a factor that could contribute to their typical aromas. Table 1 provides a complete detail about the aroma compositions of the three hop varieties, indicating that the presence of certain compounds may contribute to the differences in aroma.

Table 1. Aroma profiles of the three hop varieties, ordered by compound class then by retention time. Reported aromas are indicated.

Name Retention time (min) Peak area Aroma
‘Fuggle’ ‘Goldings’ ‘Target’
Acids
2-Methylpropanoic acid 4.818 7.92 × 105 1.78 × 105 Rancid butter3
Ketones
Butan-2-one 2.475 2.85 × 104 Chocolate, cheese, butter, ethereal, gas1
Methyl isopropyl ketone 3.057 6.58 × 104 Sweet4
Acetoin 3.821 1.29 × 105 1.36 × 105 Dairy sweet, buttery1
Methyl isobutyl ketone 4.495 1.66 × 105 Sharp, solvent-like with green, herbal, fruity1
Nonan-2-one 17.455 2.47 × 106 1.02 × 106 2.01 × 106 Varnish4
Decan-2-one 21.207 1.26 × 106 2.89 × 105 2.73 × 106 Citrus, orange-like1
Undecan-2-one 24.743 5.68 × 106 4.32 × 106 1.56 × 107 Fruity, musty, dusty, green1
Tridecan-2-one 31.178 2.07 × 106 Spicy, herbaceous1
Sulfur compounds
Dimethyl disulfide 4.555 1.09 × 104 Sulfury, cabbage, putrid1
S-Methyl 2-methylpropanethioate 7.865 2.97 × 105 Cheesy, estery, cooked vegetable5
S-Methyl 3-methylbutanethioate 11.328 8.90 × 105 Cheesy, estery, cooked vegetable5
Monoterpenes
α-Pinene 11.025 3.75 × 105 3.49 × 106 Terpenic1
Camphene 11.590 3.35 × 105 5.35 × 105 3.04 × 106 Pine, oily, herbal1
β-Pinene 12.719 1.04 × 106 1.22 × 106 1.40 × 107 Musty, green, sweet, pine1
α-Myrcene 12.938 4.41 × 105 8.12 × 105 5.38 × 107
β-Myrcene 13.442 2.83 × 108 3.56 × 108 9.38 × 108 Musty, sweet, lemon, spicy, woody1
α-Phellandrene 13.870 1.70 × 105 Terpenic, citrus, lime, green3
α-Thujene 13.888 5.89 × 106
Sylvestrene 14.844 5.24 × 106 1.35 × 107 6.55 × 107
trans-β-Ocimene 15.328 4.75 × 105 1.01 × 106 1.65 × 107 Green, tropical, woody, floral3
α-Ocimene 15.721 2.22 × 106 6.02 × 106 5.96 × 107
γ-Terpinene 16.068 3.74 × 105 4.95 × 105 6.26 × 106 Citrus-like, herbaceous, fruity, sweet1
δ-Terpinene (Terpinolene) 17.205 5.15 × 105 9.14 × 105 1.20 × 107 Sweet, pine, citrus3
Linalool 17.719 1.53 × 106 5.89 × 106 8.33 × 106 Green, floral, lemon, lavender1
Sesquiterpenes
Ylangene 27.247 3.37 × 105 7.41 × 105 1.85 × 106 Fruity1
α-Copaene 27.395 2.27 × 106 4.39 × 106 9.06 × 106 Woody, earthy1
Caryophyllene 28.784 3.77 × 107 8.24 × 107 1.05 × 108 Oily, fruity, woody1
α-Humulene 29.847 1.72 × 108 2.28 × 108 1.83 × 108 Musty, spicy, woody1
β-Farnesene 29.978 3.00 × 107 Oily, fruity, citrus-like, woody1
γ-Muurolene 30.570 1.69 × 106 2.83 × 106 1.04 × 107 Oily, herbaceous1
β-Selinene 30.853 8.14 × 105 1.10 × 106 5.98 × 106
α-Selinene 31.125 9.78 × 106 Pepper-like, orange1
γ-Cadinene 31.703 1.69 × 106 3.57 × 106 9.69 × 106
cis-Calamenene 31.971 8.30 × 105 7.26 × 105 1.53 × 106 Weak spicy, weak floral1
δ-Cadinene 31.987 5.14 × 106 7.54 × 106 1.90 × 107 Wood, herbaceous1
α-Cadinene 32.394 3.27 × 105 5.41 × 105 Dry wood, weak medicinal1
α-Calacorene 32.553 1.16 × 105 3.25 × 105 Woody, fruity, sweet, pine1
Caryophyllenyl alcohol 33.369 4.79 × 105 2.94 × 105 2.33 × 105 Warm, moss-like, spicy3
Esters
2-Methylpropyl propanoate 8.242 1.43 × 105 Sweet, fruity, bitter3
2-Methylbutyl acetate 9.110 1.46 × 105 1.86 × 105 1.57 × 106 Herbaceous, ethereal, rum, fruity1
2-Methylpropyl 2-methylpropanoate 10.453 3.93 × 106 4.71 × 104 3.97 × 107 Pineapple3
3-Methylbutyl 2-methylpropanoate 14.372 2.00 × 106 5.98 × 107
2-Methylbutyl 2-methylpropanoate 14.512 2.14 × 107 6.82 × 107
Methyl octanoate 18.705 8.59 × 105 1.63 × 106 2.98 × 106 Orange, fruity, green1
2-Methylbutyl 3-methylbutanoate 18.089 1.48 × 106 1.54 × 106 Herbaceous, fruity, sweet3
Ethyl octanoate 21.397 2.90 × 105 Fruity, floral, apricot-like3
Methyl nonanoate 22.342 1.90 × 105 5.00 × 105 1.46 × 106 Fruity, nut-like, coconut-like1
Methyl geranate 25.733 1.84 × 105 2.87 × 105 2.95 × 106 Green, fruit, floral1
Octyl 2-methylpropanoate 26.468 7.99 × 104 Fruity, fatty, grape3
Alcohols
2-Methylpropan-1-ol 2.702 1.25 × 105 8.06 × 104 Disagreeable, wine-like3
3-Methylbutan-2-ol 3.323 1.12 × 105 Fruity, fresh3
3-Methylbutan-1-ol 4.344 9.80 × 105 1.29 × 105 Whisky, pungent, balsamic, alcohol1
2-Methylbutan-1-ol 4.436 1.16 × 106 5.33 × 106 4.10 × 105 Malty, balsamic, wine, ripe onion1
Pentan-1-ol 5.287 1.49 × 105 Fruity, green, sweet, pungent1
(Z)-Hex-3-en-1-ol 8.189 4.02 × 105 1.33 × 106 1.77 × 105 Green, herbaceous3
Benzyl alcohol 15.094 5.10 × 105 Aromatic, floral, fruity1

Some prominent differences are given below:

  • Target contained a large amount of the Undecan-2-one compound. This matches with the findings presented by Collin and Lermusieau, where the high abundance of this compound helped in distinguishing Target from other European hops.

  • S-methyl 3-methylbutanethioate and S-methyl 2-methylpropanethioate are sulfur-containing compounds. These were found only in the Target variety, and can possibly impart a disagreeable cheese-like or cooked vegetable aroma.

  • Both Target and Fuggle contained 2-Methylpropanol, giving an undesirable wine-like aroma.

Increased Confidence with Select-eV

While BenchTOF systems can provide excellent spectral quality, detection of separate terpenoids, due to similar spectra and weak molecular ions, would still be difficult if standard ionization is used.

To resolve this issue, fresh sorbent tubes were used to collect the split flowing from TD analysis of individual samples and the analysis was repeated by utilizing Select-eV soft ionization at 12eV. Figure 4 shows a selection of spectral comparisons.

Spectral comparisons at 70 and 12 eV for a selection of the mono- and sesquiterpenoids that are important for contributing the characteristic aroma of hops to beer.

Spectral comparisons at 70 and 12 eV for a selection of the mono- and sesquiterpenoids that are important for contributing the characteristic aroma of hops to beer.

Spectral comparisons at 70 and 12 eV for a selection of the mono- and sesquiterpenoids that are important for contributing the characteristic aroma of hops to beer.

Spectral comparisons at 70 and 12 eV for a selection of the mono- and sesquiterpenoids that are important for contributing the characteristic aroma of hops to beer.

Figure 4. Spectral comparisons at 70 and 12 eV for a selection of the mono- and sesquiterpenoids that are important for contributing the characteristic aroma of hops to beer.

Soft ionization not only provided strong intensity for the molecular ions, but also reduced the degree of fragmentation. This resulted in spectra that were both simple and more selective. The increased intensity for the diagnostic ions offers better limits of detection and more confidence in the trace-level detection of identical components. Select-eV also retains some amount of fragmentation, thus enabling easy library-matching and assisting structural description, unlike other soft ionization methods.

Conclusion

The TD–GC–TOF MS can be effectively used for studying hops and other strong aromatic plant materials. Markes’ BenchTOF system provides reference-quality spectra that facilitated detailed characterization of VOCs in the three varieties of hops.

Select-eV allows confident identification of mono- and sesquiterpenes and other similar complex analytes. These high-performance capabilities help in making fast and robust comparison between the samples, ultimately making this method useful for robust quality control in the brewing sector.

References

  1. "A powerful methodological approach combining headspace solid phase microextraction, mass spectrometry and multivariate analysis for profiling the volatile metabolomic pattern of beer starting raw materials", J.L. Gonçalves et al, Food Chem, 2014.

  2. "Characterization of novel single-variety oxygenated sesquiterpenoid hop oil fractions via headspace solidphase microextraction and gas chromatography-mass spectrometry/olfactometry", F. Van Opstaele et al, J. Agr Food Chem, 2013.

  3. "Fenaroli’s Handbook of Flavor Ingredients", G.A. Burdock, CRC Press (5th Ed), 2004

  4. "Hop essential oil: Analysis, chemical composition and odor characteristics", G. Eyres and J. Dufour, Ch. 22 in "Beer in Health and Disease Prevention", ed. V.R. Preedy, Academic Press, 2009.

  5. "Volatile organosulphur compounds in hops and hop oils: A review", T.L. Peppard, J I Brewing, 1981.

  6. "Hop aroma extraction and analysis", G. Lermusieau and S. Collin, Ch. 5 in "Analysis of Taste and Aroma (Molecular Methods of Plant Analysis, vol. 21)", ed. J.F. Jackson and H.F. Linskens, Springer, 2002

This information has been sourced, reviewed and adapted from materials provided by Markes International Limited.

For more information on this source, please visit Markes International Limited.

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