Quantitation Method for Polyfunctional Thiols in Hops (Humulus

Subscriber access provided by AUSTRALIAN NATIONAL UNIV

Article

A New Quantitation Method for Polyfunctional Thiols in Hops (Humulus lupulus L.) and Beer using Specific Extraction of Thiols and Gas Chromatography-Tandem Mass Spectrometry Koji Takazumi, Kiyoshi Takoi, Koichiro Koie, and Youichi Tuchiya Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.7b02996 • Publication Date (Web): 03 Oct 2017 Downloaded from http://pubs.acs.org on October 8, 2017

Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a free service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are accessible to all readers and citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.

Analytical Chemistry is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

Page 1 of 19

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Analytical Chemistry

A New Quantitation Method for Polyfunctional Thiols in Hops (Humulus lupulus L.) and Beer using Specific Extraction of Thiols and Gas Chromatography-Tandem Mass Spectrometry Koji Takazumi*1, Kiyoshi Takoi2, Koichiro Koie3, Youichi Tuchiya1 1

Frontier Laboratories for Value Creation, Sapporo Holdings Ltd., 10 Okatohme, Yaizu, Shizuoka, 425-

0013, Japan 2

Product & Technology Innovation Department, Sapporo Breweries Ltd., 10 Okatohme, Yaizu,

Shizuoka, 425-0013, Japan 3

Bioresources Research & Development Department, Sapporo Breweries Ltd., 3-5-25 Kamifurano-cho

Motomachi, Sorachi-gun, Hokkaido, 071-0551, Japan *Corresponding author (e-mail [email protected]; fax +81-54-629-3144). The authors declare no competing financial interest.

Abstract A method for the quantitation of six polyfunctional thiols, 4-methyl-4-sulfanylpentan-2-one (4MSP), 3sulfanyl-4-methylpentan-1-ol (3S4MP), 3-sulfanyl-4-methylpentyl acetate (3S4MPA), 3-sulfanyl-3methylbutan-1-ol (3S3MB), 3-sulfanylhexan-1-ol (3SH), and 3-sulfanylhexyl acetate (3SHA), in hops and beer without organic mercury compounds was developed. The method employed specific extraction of thiols using a silver ion solid phase extraction (SPE) cartridge and gas chromatography-tandem mass spectrometry (GC-MS/MS). For all thiols analyzed, good linearity was achieved by adding thioglycerol ACS Paragon Plus Environment


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 2 of 19

as an analyte protectant. Recoveries for both hops (74–100%) and beer (79–113%) were acceptable, and the repeatability for both was also good (relative standard deviations of 2.8–8.4%). The limits of detection for the six polyfunctional thiols were below their odor thresholds in beer. The method was applied to quantitation of hops and beer flavored with thiol-containing hop varieties. Due to their detected levels and level variations in different beers, 4MSP and 3S4MP are thought to be important polyfunctional thiols for the characteristic flavor of hop varieties.

Polyfunctional thiols are important compounds for the aroma of food and beverages because of their very low odor thresholds and characteristic odors.1 In the field of wine research, there are many studies on polyfunctional thiols, and it is well known that 3-sulfanylhexan-1-ol (3SH), 3-sulfanylhexyl acetate (3SHA), and 4-methyl-4-sulfanylpentan-2-one (4MSP) contribute to the characteristic flavors of white wines made from several grape varieties, such as Sauvignon Blanc, Gewürztraminer, Riesling, Muscat, Petit Manseng, and Colombard.2 Recently, beer research has also begun to focus on polyfunctional thiols derived from hops (Humulus lupulus L.). Steinhaus et al.3 and Kishimoto et al.4 revealed that 4MSP contributes to the characteristic flavor of Cascade hops. Kishimoto et al.5 reported on the behavior of 3SH and 3SHA during the brewing process. Other polyfunctional thiols have also been investigated in hops and beer. Takoi et al.6 revealed that 3-sulfanyl-4-methylpentan-1-ol (3S4MP) and 3-sulfanyl-4-methylpentyl acetate (3S4MPA) contribute to the Sauvignon-like flavor of Nelson Sauvin hops. Gros et al.7 reported that 41 polyfunctional thiols have been found in Tomahawk hops. The analysis of polyfunctional thiols is a challenging task because of their low concentration and high reactivity. Several methods to analyze ultra-trace amounts of polyfunctional thiols have been developed. The most commonly used method is the specific extraction of thiols using organic mercury compounds.8,9 Kishimoto et al.4,5,10 employed the specific extraction of thiols with phydroxymercurybenzoate for the quantification of 4MSP, 3SH, and 3SHA in hops and beer. Reglitz et al.11 used mercurated agarose gel for the quantification of 4MSP in hops. These methods have good

ACS Paragon Plus Environment

Page 3 of 19

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


enough sensitivity, and they can be coupled to olfactometry to identify odor-active thiols. However, they require numerous complicated steps and use very harmful organic mercury compounds. Another quantitation approach involves the derivatization of target analytes, for which various derivatization reagents have been reported.12–15 Mateo-Vivaracho et al.13 reported derivatization by 2,3,4,5,6-pentafluorobenzyl bromide on a solid phase extraction (SPE) cartridge coupled to a gas chromatography-negative chemical ionization/mass spectrometer for the quantitation of five polyfunctional thiols in wine. Ochiai et al.14 employed derivatization by ethyl propiolate and stir bar sorptive extraction-thermal desorption-gas chromatography-tandem mass spectrometry (GC-MS/MS) for the quantitation of 4MSP, 3SH, and 3SHA in hop water extract, wort, and beer. Both methods have sufficient sensitivity and are relatively simple; however, derivatization methods are difficult to adapt for qualitative analysis and cannot be coupled to olfactometry. In this study, we developed a new specific extraction method for thiols without the use of organic mercury compounds. As it is well known that metal ions exhibit a high affinity to thiols, we used a silver ion SPE cartridge instead of mercury compounds, and coupled the specific extraction method to GC-MS/MS for the quantitation of six polyfunctional thiols (4MSP, 3S4MP, 3S4MPA, 3-sulfanyl-3methylbutan-1-ol (3S3MB), 3SH, and 3SHA) (see Fig. 1) in hops and beer.

Experimental Section Reagents and other materials 4MSP was purchased from Combi-Blocks (San Diego, CA). 3SH was purchased from Alfa Aesar (Lancashire, United Kingdom). 3SHA was purchased from Matrix Scientific (Columbia, SC). 3S3MB and 5,5’-dithio-bis-(2-nitrobenzoic acid) were purchased from Tokyo Chemical Industry (Tokyo, Japan). 3-Sulfanylpropyl hexanoate (3SPH) and thioglycerol were purchased from Wako Pure Chemical Industries (Osaka, Japan). 3S4MP and 3S4MPA were synthesized as described in a previous study.6 The MetaSep IC-Ag SPE column was purchased from GL sciences (Tokyo, Japan).



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 4 of 19

Hop samples Hallertauer Tradition hops were purchased from Joh. Barth & Sohn (Nuremberg, Germany). Hallertau Blanc and Mandarina Bavaria were provided by HVG (Wolnzach, Germany). Cascade and Zeus were provided by John I. Haas (Yakima, WA). Citra and Ekuanot were provided by Yakima Chief (Sunnyside, WA). Galaxy and Vic Secret were provided by Hop Products Australia (Tasmania, Australia), and Nelson Sauvin was provided by New Zealand Hops Limited (Nelson, New Zealand). Cascade, Zeus, Citra, Ekuanot, and Nelson Sauvin hops were provided as dry hop cones. Others were pellets. All hops were milled with a cutter mill (ZM200, Verder Scientific Co., Ltd., Germany) at 15,000 rpm with a 0.5 mm mesh and stored at -20 °C until required for analysis and brewing.

Brewing process Beers were made using cooled wort prepared in a factory (Shizuoka Brewery, Sapporo Breweries, Ltd.). The cooled wort was prepared using commercially available malts and hops without flavoring. For hop flavoring, 4 g of hops (Hallertauer Tradition, Hallertau Blanc, Mandarina Bavaria, Cascade, Zeus, Citra, Ekuanot, Galaxy, Vic Secret, or Nelson Sauvin) was added to a bottle containing 200 mL of the cooled wort and autoclaved at 105 °C for 5 min. After cooling, each hop-flavored wort was mixed with 2300 mL of cooled wort in European Brewery Convention (EBC) tall tubes.16 These conditions corresponded to that of late-hopping, with a hop concentration of 1.6 g/L. The fermentation was started by adding 10.0 × 106 cells/mL lager yeast (brewery collected; Saccharomyces pastorianus) to the mixed wort and carried out at 12 °C for 5 days. After maturation, the finished beer was obtained by centrifuging at 12,000 g for 10 min.

Specific extraction of thiols from hops and beer Extraction of thiols from hop samples


Page 5 of 19

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


In a conical flask, 2 g of milled pellets, 100 mL of dichloromethane, and 20 µL internal standard solution (100 mg/L 3-sulfanylpropyl hexanoate in ethyl acetate) were stirred with a magnetic stirrer. After 30 min, the mixture was filtered with a glass filter.

Extraction of thiols from beer To a 50 mL glass centrifuge tube, 6 g of NaCl, 20 mL of beer, 20 mL of dichloromethane, and 20 µL of internal standard solution (10 mg/L 3-sulfanylpropyl hexanoate in ethanol) were added and shaken for 15 min. After centrifugation at 1800 g for 15 min, the organic phase was obtained.

Specific extraction of thiols from hop and beer extracts SPE was carried out with an ASPEC GX-274 (Gilson, Middleton, WI). First, an SPE cartridge (MetaSep IC-Ag) was conditioned with 6 mL of dichloromethane. Then, 10 mL of hop extract or all the beer extract was loaded onto the cartridge at a flow rate of 2 mL/min. The cartridge was then rinsed with 10 mL of dichloromethane and 20 mL of acetonitrile in succession. The cartridge was reversed and washed with 10 mL of dichloromethane. Polyfunctional thiols were eluted with 6 mL of 10 g/L thioglycerol in dichloromethane at a flow rate of 0.66 mL/min. The eluate and 30 mL of saturated salt solution were added to a 50 mL glass centrifuge tube and shaken for 15 min. After centrifugation at 1800 g for 15 min, the organic phase was obtained and dried on anhydrous sodium sulfate. Finally, 1 mL of ethyl acetate was added to the eluate, and the mixture was concentrated to 20 µL under nitrogen flow.

Calibration curves Calibration curves were obtained by analyzing standard mixture solutions. The concentration of each polyfunctional thiol stock solution was determined according to Ellmans’s method17 using 5,5dithiobis(2-nitrobenzoic acid) before mixing. Standard mixture solutions were prepared by mixing six polyfunctional thiol stock solutions and diluting them with ethyl acetate. To all standard mixture



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 6 of 19

solutions, 3-sulfanylpropyl hexanoate (final concentration 10 mg/L) and thioglycerol (final concentration 10 g/L) were added as an internal standard and an analyte protectant, respectively.

Validation of the method Validation was carried out using Hallertauer Tradition hop pellets and a commercial pilsner type beer that contained 537 ng/L of 3S3MB and 47 ng/L of 3SH. Recovery, limit of detection (LOD) and limit of quantitation (LOQ) were obtained as the average of duplicate analyses, while repeatability was determined by six replicate analyses. The LOD and LOQ corresponded to the concentration of analytes that gave a three and ten times higher signal than noise, respectively. Noise was defined as 3 times rootmean-square .

Gas chromatography-tandem mass spectrometry GC-MS/MS analysis was performed with an Agilent 7890A gas chromatograph coupled to a 7000B triple quadrupole mass spectrometer (Agilent Technologies, Palo Alto, CA). An InertCap PureWAX capillary column (30 m × 0.25 mm inner diameter, 0.25 µm film thickness; GL Sciences) was used for separation. Here, 3 µL of extract was injected using a CombiPal system (CTC Analytics, Zwingen, Switzerland). The inlet was operated in pulsed splitless mode (30 psi, 1 min) at 250 °C. The flow rate of the helium carrier gas was 1 mL/min. The oven temperature was increased from 70 °C (held for 1 min) to 250 °C at a rate of 5 °C/min, and was held at 250 °C for 10 min. The triple quadrupole mass spectrometer was operated in selected reaction monitoring (SRM) mode. Target and qualification transitions and the corresponding collision energy values are listed in Table 1.

Results and Discussion Calibration curve Calibration curves were obtained by analyzing nine standard solutions. The linearity of the internal standard calibration curves was evaluated. The peak areas of target compounds obtained from the low ACS Paragon Plus Environment

Page 7 of 19

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


concentration standard solution were lower than the calculated areas from the calibration curves. This is due to interactions of the analytes with the active sites in the GC inlet. It is well known that a matrix of sample can mask the active sites in the inlet and increase the transfer of analyte to the column.18 One way to improve the linearity is to use matrix-matched calibration standards, which increases the number of samples to be prepared. Thus, the use of an analyte protectant was examined. To examine its effectiveness, a 2.5 µg/L standard mixture was prepared at three concentrations (0, 5, and 10 g/L) of thioglycerol. The responses for all the thiols were increased in the presence of thioglycerol, and more than 5 g/L of thioglycerol provided the largest response (Fig. 2). Therefore, thioglycerol was added to all standard mixture solutions to make a 10 g/L final concentration. As a result, good linearity with r2 ≥ 0.993 was achieved for all compounds analyzed without specific treatment of the inlet and liner for deactivation. Typical calibration curves and calibration curve parameters are shown in Fig. 3 and Table 2.

Optimization of the specific extraction of thiols In the field of wine research, specific extraction methods for thiols have been developed.8,9 These methods use the interaction of thiols with organic mercury compounds. The originality of the method in this paper lies in the use of silver ions instead of harmful organic mercury compounds for the extraction of thiols. Also, our method is advantageous in that it uses commercially available silver ion SPE cartridges and an automated SPE instrument. The SPE procedure was optimized using hop samples. Optimization was carried out for the sample loading, column washing, and analyte elution steps. In some methods,5,9,19 wine or beer samples were directly loaded onto the column. Beer samples could not be directly loaded onto the silver ion SPE cartridge because the cartridge became blocked. One of the common uses of silver ion SPE cartridges is for the separation of the methyl esters of fatty acids. For this purpose, samples were dissolved in dichloromethane.20 Therefore, liquid-liquid extraction could be conducted, and the cartridge was not blocked. ACS Paragon Plus Environment


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 8 of 19

The washing step was optimized by considering the recoveries and washing effects. Three washing solvents—methanol, acetonitrile, and water—were evaluated. The choice of solvent did not affect the recovery; however, the interfering matrix components were less when acetonitrile was used. In the elution step, two elution solutions, 10 g/L cysteine in water and 10 g/L thioglycerol in dichloromethane, were evaluated. When eluting with 10 g/L thioglycerol in dichloromethane, the recovery of hydrophobic thiols such as 3MHA improved. After SPE, most of thioglycerol was removed by washing with saturated salt solution. The residual thioglycerol level was adjusted to 3.3–5 mg/L by varying the volume of salt solution used. After concentration, the final extract contained 10–15 g/L of thioglycerol, which served as an analyte protectant. Figure 4 shows the total ion chromatogram obtained from the hop extract (A) before and (B) after the specific extraction of thiols.

Validation of the method Validation of the method was conducted in terms of recovery, repeatability, LOD and LOQ. These parameters were evaluated for both hops and beer. The recovery for hops (74–100%) and beer (79– 113%) are within an acceptable range. Repeatability, expressed as the relative standard deviation (RSD), was determined using six replicate analyses. The repeatability results (RSD of 2.8–6.3% for hops and 3.3–8.4% for beer) were also satisfactory. The LOD and LOQ were determined by analyzing low-level spiked or non-spiked samples. For all analytes, the LOD for beer was below their respective odor thresholds. A typical SRM chromatogram of beer is shown in Fig. 5, and the results of validation are summarized in Table 3.

Application to hop and beer analysis Ten beer varieties, hopped with different kinds of hops, were brewed, and our validated method was applied to analyze these hops and beer. Table 4 shows the results of the hop quantitation. Except for 3SHA, five polyfunctional thiols were detected in hops. The detection ranges were very wide. The ACS Paragon Plus Environment

Page 9 of 19

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


difference between the maximum and minimum concentration values was more than 20-fold for all detected polyfunctional thiols. There were no correlations between each pair of polyfunctional thiols. The amount of 4MSP was very low in all German varieties, while high amounts were found in American, New Zealand, and Australian varieties, as described in previous papers.10,11 On the other hand, such trends were not observed for other polyfunctional thiols. Reglitz et al.11 reported that the 4MSP content in Citra was the highest among 45 varieties. Cibaka et al.19 reported that the 3S4MP content in Hallertau Blanc was high. These results were in good agreement with our results. Table 5 shows the results for the beer quantitation. Five polyfunctional thiols were detected in beer, although the 3SH and 3S3MB content did not differ much among hop varieties. In several beers, the 4MSP, 3S3MB, 3S4MP, and 3SH contents were higher than their respective odor thresholds. It is thought that 4MSP and 3S4MP are important polyfunctional thiols for the characteristic flavor of hop varieties because of their detection levels and level variations in different beers. The origin of thiols in beer is a major question. In wine research, it is well known that 3SH is released from cysteine and glutathione conjugates by yeast during fermentation.22–25 Figure 6 shows the relationships between the polyfunctional thiol content in hops and beer. A linear correlation was found for 4MSP and 3S4MP. According to this result, the 4MSP and 3S4MP content in beer seems to be determined by the free thiols contained in hops. In contrast, no correlation was found for 3S3MB and 3SH. For these thiols, the detected content in beer was much higher than the value calculated from the hopping rate and the free thiol content in hops by assuming that the transferring ratio from hops to beer is 100% (Table 6). These results indicate that the free thiols contained in hops are not the major origin of 3S3MB and 3SH in beer.

Conclusions A new quantitation method for polyfunctional thiols has been developed, validated, and applied to hops and beer analysis. We employed a specific extraction of thiols using a silver ion SPE cartridge instead of



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 10 of 19

harmful organic mercury compounds. The method involved a relatively simple separation procedure using an automated SPE instrument. Compared to other specific extraction methods, the LOD of 4MSP was superior with our method and was higher than its odor threshold in beer or white wine. Other analytical parameters were at least equivalent to previously reported methods, which are more complicated, require more sample, and use dangerous organic mercury compounds. Some reported derivatization methods involve simpler procedures that do not require organic mercury compounds. Repeatability and recovery of these methods were slightly superior to those of our method because these methods use stable isotope-labeled internal standards. However, our method exhibits good analytical performance similar to these methods without the need for isotope-labeled internal standards. Therefore, our method can be used for the analysis of minor thiols for which stable isotope-labeled internal standards cannot be obtained. Furthermore, the literature methods can only be adapted for aqueous samples, while our method may be applied to both solid and aqueous matrices other than hops and beer. Compared to derivatization methods, one advantageous aspect of the specific extraction method is its applicability for the qualitative analysis of thiols, which will be demonstrated in a later study.

References (1) Blank, I. Sensory Relevance of Volatile Organic Sulfur Compounds. An overview. In Heteroatomic Aroma Compounds; Reineccius, G. A., Reineccius, T. A., Eds.; American Chemical Society (ACS): Washington, D.C., 2002; ACS Symposium Series, Vol. 826, Chapter 2, pp 25–53. (2) Tominaga, T.; Baltenweck-Guyot, R.; Peyrot Des Gachons, C.; Dubourdieu, D. Am. J. Enol. Vitic. 2000, 51, 178–181. (3) Steinhaus, M.; Wilhelm, W.; Schieberle, P. Eur. Food. Res. Technol. 2007, 226, 45–55. (4) Kishimoto, T.; Wanikawa, A.; Kono, K.; Shibata, K. J. Agric. Food Chem. 2006, 54, 8855–8861. ACS Paragon Plus Environment

Page 11 of 19

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


(5) Kishimoto, T.; Morimoto, M.; Kobayashi, M.; Yako, N.; Wanikawa, J. Am. Soc. Brew. Chem. 2008, 3, 192−196. (6) Takoi, K.; Degueil, M.; Shinkaruk, S.; Thibon, C.; Maeda, K.; Ito, K.; Bennetau, B.; Dubourdieu, D.; Tominaga, T. J. Agric. Food Chem. 2009, 57, 2493−2502. (7) Gros, J.; Nizet, S.; Collin, S. J. Agric. Food Chem. 2011, 59, 8853−8865. (8) Tominaga, T.; Murat, M. L.; Dubourdieu, D. J. Agric. Food Chem. 1998, 46, 1044–1048. (9) Tominaga, T.; Dubourdieu, D. J. Agric. Food Chem. 2006, 54, 29−33. (10) Kishimoto, T.; Kobayashi, M.; Yako, N.; Iida, A.; Wanikawa, A. J. Agric. Food Chem. 2008, 56, 1051–1057. (11) Reglitz, K.; Steinhaus, M. J. Agric. Food Chem. 2017, 64, 2364–2372. (12)Mateo-Vivaracho, L.; Cacho, J.; Ferreira, V. J. Chromatogr. A 2007, 1146, 242−250. (13) Mateo-Vivaracho, L.; Cacho, J.; Ferreira, V. J. Chromatogr. A 2008, 1185, 9−18. (14) Ochiai, N.; Sasamoto, K.; Kishimoto, T. J. Agric. Food Chem. 2015, 63, 6698–6706. (15) Capone, D. L.; Ristic, R.; Pardon, K. H.; Jeffery, D. W. Anal. Chem. 2015, 87, 1226−1231. (16) EBC Analytica Microbiologica. J. Inst. Brew. 1977, 83, 109-118. (17) Ellman, G. L. Tissue sulfhydryl groups. Arch. Biochem. Biophys. 1959, 82, 70–77. (18) Poole, C. F. J. Chromatogr. A 2007, 1158, 241−250. (19) Mateo-Vivaracho, L.; Cacho, J.; Ferreira, V. J. Sep. Sci. 2009, 32, 3845–3853 (20) Christie, W. W. J. Lipid Res. 1989, 30, 1471−1473.



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 12 of 19

(21) Kankolongo Cibaka, M. L.; Gros, J.; Nizet, S.; Collin, S. J. Agric. Food Chem. 2015, 63, 3022−3030. (22) Peyrot des Gachons, C.; Tominaga, T.; Dubourdieu, D. J. Agric. Food Chem. 2000, 48, 3387−3391. (23) Subileau, M.; Schneider, R.; Salmon, J. M.; Degryse, E. J. Agric. Food Chem. 2008, 56, 9230−9235. (24) Capone, D. L.; Sefton, M. A.; Hayasaka, Y.; Jeffery, D.W. J. Agric. Food Chem. 2010, 58, 1390−1395. (25) Winter, G.; Van Der Westhuizen, T.; Higgins, V. J.; Curtin, C.; Ugliano, M. Aust. J. Grape Wine Res. 2011, 17, 285−290.

Acknowledgments We thank Mako Yabe at the Frontier Laboratories for Value Creation for technical assistance.

Figures and tables

Figure 1. Chemical structures of polyfunctional thiols


Page 13 of 19

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Figure 2. Comparison of normalized peak areas of polyfunctional thiols in standard solutions as a function of thioglycerol concentration

Figure 3. Typical calibration curves



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 14 of 19

Figure 4. Total ion chromatogram of Hallertauer Tradition hop extract (A) before and (B) after specific extraction of thiols with a silver ion SPE cartridge. Both extracts were concentrated ten times

Figure 5. SRM chromatograms obtained by analyzing spiked or non-spiked beer. (A) 4MSP, spiked at 10 ng/L; (B) 3S3MB, non-spiked; (C) 3S4MPA (left peak) and 3SHA (right peak), spiked at 25 ng/L; (D) 3S4MP, spiked at 25 ng/L; (E) 3SH, non-spiked ACS Paragon Plus Environment

Page 15 of 19

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Figure 6. Relationships between polyfunctional thiol contents in hops and beer

Table 1. SRM conditions for polyfunctional thiols



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 16 of 19

target

qualification

compound

retention time (min)

precursor ion

product ion

collision energy (v)

precursor ion

product ion

collision energy (v)

4MSP

7.74

132

89

6

132

75

2

3S3MB

12.89

120

71

10

86

71

6

3S4MPA

13.99

116

88

4

116

101

4

3SHA

14.27

116

88

4

116

101

4

3S4MP

16.48

134

100

0

134

57

8

3SH

16.80

134

82

2

100

82

0

3SPH

17.76

106

60

4

106

88

0

Table 2. Calibration curve parameters hop

beer 2

2

compound

range (µg/kg)

points

R

range (ng/L)

points

R

4MSP

0–100

9

0.999

0–100

6

0.998

3S3MB

0–50

8

0.998

0–1000

9

0.999

3S4MPA

0–100

9

0.999

0–100

6

0.997

3SHA

0–50

8

0.998

0–100

6

0.997

3S4MP

0–1000

9

0.999

0–1000

6

0.993

3SH

0–50

8

0.996

0–250

7

0.996

Table 3. Recovery, RSD, LOD and LOQ of polyfunctional thiols in hops and beer hop

a

beer odor threshold (ng/L)f

compound

recovery (%)a

RSD (%)a

LOD (µg/L)b

LOQ (µg/L)b

recovery (%)c

RSD (%)d

LOD (ng/L)e

LOQ (µg/L)b

4MSP

73

6.3

0.2

0.6

79

6.8

1.4

4.6

1.5

3S3MB

83

5.0

0.4

1.2

110

7.8

18.8

62.8

1500

3S4MPA

95

2.8

0.3

1.1

108

3.3

3.8

12.7

160

3SHA

100

3.2

0.5

1.7

110

5.3

3.7

12.5

4

3S4MP

97

5.3

0.7

2.3

76

8.4

7.1

23.7

70

3SH

93

4.1

0.1

0.2

113

6.2

1.9

6.3

55

(4) (8)

(6)

(5) (6) (4)

Recovery and repeatability were determined using spiked hop samples with 25 µg/kg of the six

polyfunctional thiols. b LODs and LOQs were determined using spiked hop samples with 1 µg/kg of the six polyfunctional thiols. c Recoveries were determined using beer spiked with 10 ng/L 4MSP, 100 ng/L


Page 17 of 19

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


3S3MB, 25 ng/L 3S4MPA, 25 ng/L 3SHA, 25 ng/L 3S4MP, and 100 ng/L 3SH. d Repeatability was determined using spiked (10 ng/L of 4MSP, 3S4MPA, 3SHA, and 3S4MP) or non-spiked (3S3MB and 3SH) beer. e LODs were determined using spiked (5 ng/L 4MSP, 25 ng/L 3S4MPA, 5 ng/L 3SHA, and 25 ng/L 3S4MP) or non-spiked (3S3MB and 3SH) beer. f Odor thresholds are in beer except for 3S3MB, which was determined in a model wine.

Table 4. Polyfunctional thiol content in various hop varieties 4MSP

a

3S3MB

a

3S4MPA

a

a

3SHA

3S4MP a

3SH

variety

country

(µg/kg)

(µg/kg)

(µg/kg)

(µg/kg)

(µg/kg)

(µg/kg)a

Hallertauer Tradition

Germany

1.6

7

1.1

Germany

nd c 195

nd

Hallertau Blanc

trb 2.8

nd

295

11.8

Mandarina Bavaria

Germany

1.1

3.4

4

nd

61

5.7

Cascade

USA

1.3

tr

1

nd

6

1.4

Zeus

USA

7.3

3.2

2

nd

5

0.9

Citra

USA

36.6

10.3

tr

nd

24

8.6

8.2

a

Ekuanot

USA

7.2

31.0

6

nd

282

23.2

Galaxy

Australia

12.7

5.5

tr

nd

26

5.7

Vic Secret

Australia

18.8

7.1

29

nd

58

2.8

Nelson Sauvin

New Zealand

22.4

36.4

61

nd

305

6.2

Values reported as the mean of duplicate analyses; b detected below the LOQ; c not detected

Table 5. Polyfunctional thiol content in different beer varieties 3S3MB

a

a

3S4MPA a

3SHA

3S4MP

a

a

3SH

variety

(ng/L)

(µg/L)

(ng/L)

(ng/L)

(ng/L)

(ng/L)a


1.53

nd

nd

28

117

Hallertau Blanc

nd b nd

1.56

16.1

nd

230

115

Mandarina Bavaria

nd

1.51

nd

nd

56

109

c

nd

nd

tr

126

Cascade

a

4MSP

Zeus

tr 5.7

1.50 1.61

nd

nd

24

106

Citra

18.6

1.77

tr

nd

45

132

Ekuanot

4.8

1.17

8.3

nd

225

100

Galaxy

5.6

1.38

nd

nd

36

121

Vic Secret

8.8

1.46

nd

nd

51

94

Nelson Sauvin

12.0

1.70

tr

nd

217

105

Values reported as the mean of duplicate analyses; b not detected; c detected below the LOQ ACS Paragon Plus Environment


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 18 of 19

Table 6. Polyfunctional thiol content calculated from the hopping rate and free thiol content in hops by assuming the transferring ratio from hop to beer is 100% 4MSP

3S3MB

3S4MPA

3S4MP

3SH

variety

(ng/L)

(ng/L)

(ng/L)

(ng/L)

(ng/L)


0.7

2.6

-

11

2

Hallertau Blanc

4.6

13.1

311

472

19

Mandarina Bavaria

1.7

5.4

6

98

9

Cascade

2.1

1.2

2

9

2

Zeus

11.7

5.1

3

8

1

Citra

58.6

16.5

1

38

14

Ekuanot

11.6

49.6

9

451

37

Galaxy

20.2

8.8

1

41

9

Vic Secret

30.1

11.3

46

94

4

Nelson Sauvin

35.8

58.2

97

488

10


Page 19 of 19

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


For TOC only:


Quantitation Method for Polyfunctional Thiols in Hops (Humulus

Recommend Documents