Development of a Method for the Quantitation of Three Thiols in Beer

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Development of a Method for the Quantitation of Three Thiols in Beer, Hop, and Wort Samples by Stir Bar Sorptive Extraction with in Situ Derivatization and Thermal Desorption−Gas Chromatography−Tandem Mass Spectrometry Nobuo Ochiai,*,† Kikuo Sasamoto,† and Toru Kishimoto‡ †

GERSTEL K.K., 1-3-1 Nakane, Meguro-ku, Tokyo 152-0031, Japan Asahi Breweries, Limited, 1-21 Midori 1-chome, Moriya-shi, Ibaraki 302-0106, Japan

‡

ABSTRACT: A method for analysis of hop-derived polyfunctional thiols, such as 4-sulfanyl-4-methylpentan-2-one (4S4M2Pone), 3-sulfanylhexan-1-ol (3SHol), and 3-sulfanylhexyl acetate (3SHA), in beer, hop water extract, and wort at nanogram per liter levels was developed. The method employed stir bar sorptive extraction with in situ derivatization (der−SBSE) using ethyl propiolate (ETP), followed by thermal desorption and gas chromatography−tandem mass spectrometry (TD−GC− MS/MS) with selected reaction monitoring (SRM) mode. A prior step involved structural identification of the ETP derivatives of the thiols by TD−GC−quadrupole−time-of-flight mass spectrometry with parallel sulfur chemiluminescence detection (Q−TOF−MS/SCD) after similar der−SBSE. The der−SBSE conditions of the ETP concentration, buffer concentration, salt addition, and extraction time profiles were investigated, and the performance of the method was demonstrated with spiked beer samples. The limits of detection (LODs) (0.19−27 ng/L) are below the odor threshold levels of all analytes. The apparent recoveries at 10−100 ng/L (99−101%) and the repeatabilities [relative standard deviation (RSD) of 1.3−7.2%; n = 6] are also good. The method was successfully applied to the determination of target thiols at nanogram per liter levels in three kinds of beer samples (hopped with Cascade, Citra, and Nelson Sauvin) and the corresponding hop water extracts and wort samples. There was a clear correlation between the determined values and the characteristics of citrus hop aroma for each sample. KEYWORDS: polyfunctional thiol, beer, hops, wort, stir bar sorptive extraction (SBSE), in situ derivatization, GC−MS/MS

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INTRODUCTION Polyfunctional thiols in foods and beverages have received special attention because of their extremely low odor threshold levels and high sensory impact.1 Several thiols, e.g., 4-sulfanyl-4methylpentan-2-one (4S4M2Pone), 3-sulfanylhexan-1-ol (3SHol), and 3-sulfanylhexyl acetate (3SHA), are well-known for their contributions to the fruity/citrus/tropical aroma of wine (e.g., Sauvignon Blanc).2,3 Also, these thiols significantly contribute to aroma of certain types of beer (e.g., hopped with U.S.A., New Zealand, and Australian hop cultivars),4−10 even at nanogram per liter levels. To analyze these thiols in beer, it is essential to have powerful extraction and enrichment steps before gas chromatographic (GC) analysis. Steinhaus et al. used solvent-assisted flavor evaporation (SAFE), followed by affinity chromatography, using mercurated agarose gel.7,8 Kishimoto et al.4−6 and Gros et al.9,10 employed the extraction method with p-hydroxymercurybenzoate (pHMB) and a strongly basic anion-exchange resin.2,11 The brewing industry has a significant interest in these thiols, but the problem is that conventional analytical methods are tedious, time-consuming, and labor-intensive. Moreover, these methods use a reagent containing mercury ion, and growing world concern related to environmental issues makes the disposal of mercury-ioncontaining wastes problematic. On the other hand, several wine researchers demonstrated mercury-free extraction methods, which include derivatization steps.12−14 The most successful method using 2,3,4,5,6-pentafluorobenzyl bromide (PFBBr) and © 2015 American Chemical Society

solid-phase extraction (SPE), followed by GC−negative chemical ionization/mass spectrometry (NCI/MS), made it possible to simultaneously analyze five thiols, including 4S4M2Pone, 3SHol, and 3SHA, below their odor threshold levels.14 However, the SPE method still includes additional procedures, e.g., pre-methoxymation of 4S4M2Pone to improve derivatization of the mercapto function and extensive cleanup/washing steps. Recently, Owen re-reported that the reaction of thiols with simple esters of propiolic acid is a versatile and powerful device for investigation of thiols in many areas of chemistry, biology, and biochemistry.15 Subsequently, Zacharis et al. demonstrated the potential of ethyl propiolate (ETP) as a novel derivatizing reagent for thiols.16 In 2013, Herbst-Johnstone et al. demonstrated an in situ derivatization SPE method using ETP for 4S4M2Pone, 3SHol, and 3SHA in wine.17 However, the limit of detection (LOD) of 4S4M2Pone in white wine with this approach (24.5 ng/L) is still much higher than that of the PFBBr-SPE method (0.6 ng/L).14 In the past decade, stir bar sorptive extraction (SBSE) has been widely used as a miniaturized and solvent-less sample preparation method in various areas, including food and flavor analysis.18−20 The extraction and desorption mechanism is Received: Revised: Accepted: Published: 6698

May 8, 2015 July 9, 2015 July 12, 2015 July 12, 2015 DOI: 10.1021/acs.jafc.5b02298 J. Agric. Food Chem. 2015, 63, 6698−6706

Journal of Agricultural and Food Chemistry

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similar to that of solid-phase microextraction (SPME) and allows for extraction and concentration in a single step, followed by thermal desorption of the entire extracted fraction into a GC system. These capabilities greatly simplify the sample preparation process and minimize any risk of contamination. Moreover, the enrichment factor for SBSE, which is determined by the analyte recovery in the extraction phase, is much higher than that of SPME using the same extraction phase, i.e., polydimethylsiloxane (PDMS), because of the 50−250 times larger volume of the extraction phase on the stir bar. SBSE can also be used in combination with a derivatization step similar to SPME. For polar solutes with low octanol−water distribution coefficient (Kow) values, the corresponding derivatives generally have higher Kow values, resulting in higher recovery and sensitivity.18 Also, higher molecular weights of derivatives provide higher selectivity in GC−MS analysis. In this regard, SBSE with in situ derivatization (der−SBSE) using pentafluorobenzylhydroxylamine (PFBHA), followed by thermal desorption (TD)−GC− MS, was demonstrated for the analysis of stale-flavor carbonyl compounds, including E-2-nonenal, in beer.21 In this study, we developed a simple and solvent-less method for the quantitation of hop-derived thiols (4S4M2Pone, 3SHol, and 3SHA) in the three different sample matrices of beer, hop water extract, and wort at nanogram per liter levels by der−SBSE using ETP, followed by TD−GC−tandem mass spectrometry (MS/MS). Although the ETP derivatives (thioacrylates) of thiols (4S4M2Pone-ETP, 3SHol-ETP, and 3SHA-ETP) increase both log Kow values (from 1.07−2.70 to 1.83−3.45) and molecular weights (from 132−176 to 230−274), the selectivity and sensitivity of conventional GC−single-quadrupole MS in selected ion monitoring (SIM) mode were not sufficient for real samples, which include very complex matrices. Therefore, GC−triplequadrupole (QQQ) MS with selected reaction monitoring (SRM) mode was used for both increased selectivity and sensitivity.

Article

MATERIALS AND METHODS

Chemicals. 4S4M2Pone, 3SHol, and 3SHA were purchased from Penta Manufacturing Co. (Livingston, NJ). d10-4-Sulfanyl-4-methylpentan2-one (d10-4S4M2Pone) was purchased from aromaLAB AG (Freising, Germany). d2-3-Sulfanylhexan-1-ol (d2-3SHol) was purchased from NARD Institute, Ltd. (Hyogo, Japan). ETP and Tris(hydroxymethyl)aminomethane hydrochloride (Tris−HCl) solution (1 M, pH 9.0) were purchased from Wako Pure Chemicals (Osaka, Japan). Sodium hydroxide (NaOH) solution (1 N) and ethanol (pesticide residue analysis grade) were purchased from Kanto Kagaku (Tokyo, Japan). Hops. The hop cultivars investigated were Cascade, Citra, and Nelson Sauvin. Cascade (6.4% α-acid pellets, 2013 crop) was purchased from John I. Haas, Inc. (Yakima, WA). Citra (12.8% α-acid pellets, 2013 crop) was purchased from Yakima Chief (Sunnyside, WA). Nelson Sauvin (11.2% α-acid pellets, 2013 crop) was purchased from New Zealand Hops, Ltd. (Nelson, New Zealand). Brewing Processes. Pellets of the hop cultivars Cascade, Citra, and Nelson Sauvin were used in the brewing process. For the evaluation of each cultivar, the wort from each hop cultivar was prepared to contain equal amounts of α acid. A 20 L volume of wort without hops was boiled for 60 min in a wort kettle. An amount of the hop pellet with 130 mg of α acid was then added to 1 L of hot wort (85 °C) in a glass vessel, and the wort was cooled promptly to 5 °C. The yeast was pitched at a rate of 20 × 106 cells/mL into the wort (12.5 °P), and static fermentations were carried out while stirring at 14.0 °C for 7 days in the 1 L glass vessel. The finished beer was obtained by cooling to −1 °C for 5 days and then centrifuging at 7000 rpm for 30 min. Hop Water Extract. The water extract of each hop cultivar was prepared to contain the same amount of α acid as the corresponding wort. The amount of each hop pellet containing 130 mg of α acid was added to 1 L of hot water (85 °C) to extract the water-soluble aroma components and then cooled promptly. The hop water extracts were obtained by centrifuging at 7000 rpm for 30 min at −1 °C. Stir Bar Sorptive Extraction with in Situ Derivatization (der− SBSE). Stir bars (Twister) coated with 24 μL of PDMS were obtained from GERSTEL (Mülheim an der Ruhr, Germany). For SBSE, 20 mL headspace vials with a metal screw cap containing polytetrafluoroethylene (PTFE)-coated silicon septa (GERSTEL) and a multiple-position

Figure 1. CAD mass spectrum of the cis-ETP derivative of 4S4M2Pone obtained from der−SBSE−TD−GC−Q−TOF−MS/SCD of spiked water at 10 ng/L. Mass errors from the theoretical formula are also shown in parentheses. 6699

DOI: 10.1021/acs.jafc.5b02298 J. Agric. Food Chem. 2015, 63, 6698−6706

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− − − − −

CH3CH2O(CO)CHCHSH]+ CH3CH2OH]+ CH3CH2O(CO)CHCHSH]+ CH3(CO)OCH3]+ CH3CH2OH]+

99.0797 185.0619 231.1003 101.0941 187.0762 143.1056 187.0770 229.0881 275.2797

m/z 6700

Collision energy of 2.5 V was used for cis-4S4M2Pone−ETP and cis-3SHA−ETP, and collision energy of 10 V was used for cis-3SHol−ETP. a

3.6 315.1606 C16H27O4S 3.7 303.1614 C15H27O4S 2.5 275.1299 C13H23O4S 0.51 C13H22O4S cis-3SHA−ETP

274.1232

9.3 273.1496 C14H25O3S 6.3 261.1524 C13H25O3S 4.2 C11H21O3S C11H20O3S cis-3SHol−ETP

232.1140

−5.4

233.1193

6.6 271.1343

m/z formula

C14H23O3S 4.8 259.1345

m/z formula

C13H23O3S 6.5 231.1033

m/z formula

C11H19O3S

formula

C11H18O3S

compound

m/z

4.1

MH+

mass error (ppm)

cis-4S4M2Pone−ETP

230.0963

mass error (ppm) [M + C2H5]+ M+

mass error (ppm)

GC−PCI−TOF−MS GC−EI−TOF−MS

Table 1. Mass Spectral Information for cis-ETP Derivatives of 4S4M2Pone, 3SHol, and 3SHA

[M + C3H5]+

mass error (ppm)

[MH [MH MH+ [MH [MH [MH [MH [MH MH+

fragment ion

a

GC−PCI−Q−TOF−MS

MS/MS fragment ion, MH+

− CH3CH2O(CO)CHCHSH]+ − CH3CH2OH]+

mass error (ppm)

magnetic stirrer (20 positions) from Global Change (Tokyo, Japan) were used. Prior to use, the stir bars were conditioned for 20 min at 250 °C in a flow of helium. Before der−SBSE, beer and wort samples were adjusted to pH 9 using NaOH solution (1 M). Hop water extract was adjusted to pH 9 using 5 mM Tris−HCl to give buffer capacity. A total of 10 mL of sample containing 35 mM ETP, internal standards (20 ng/L d10-4S4M2Pone and 200 ng/L d2-3SHol), and the PDMS stir bar was transferred to 20 mL headspace vials. The vial was sealed with the metal screw cap, and the PDMS stir bar was first stirred at room temperature (25 °C) for 10 min at 500 rpm for the ETP derivatization step.17 After 10 min of initial stirring, 30% NaCl was added and the vial was sealed again. To prevent damage to the glass jacket of the stir bar when dissolving NaCl, the PDMS stir bar was first attached to the inside of the metal screw cap with magnetic attraction. Then, the vial was gently shaken for a few minutes (horizontally). Finally, the PDMS stir bar was dropped to the bottom of the vial, and SBSE was performed for 180 min while stirring at 1500 rpm. After extraction, the stir bars were removed with forceps, dipped briefly in ultrapure water, dried with a lint-free tissue, and placed in a glass thermal desorption liner. The glass liner was placed in the thermal desorption unit. No further sample preparation was necessary. No carry-over was observed for concentrations up to 1000 ng/L after analysis. Reconditioning of stir bars was performed after use by soaking each consecutively in ultrapure water and acetonitrile for 1−2 h. Stir bars were then removed from the solvent and dried on a clean surface at room temperature for 1 h. Finally, the stir bars were thermally conditioned for 20 min at 250 °C in a flow of helium. Typically, the same PDMS stir bar could be used more than 50 times. Thermal Desorption−Gas Chromatography−Quadrupole Time-of-Flight Mass Spectrometry with Parallel Sulfur Chemiluminescence Detection (TD−GC−Q−TOF−MS/SCD). For the identification of the ETP derivatives of thiols, the TD−GC−Q− TOF−MS/SCD analysis was performed with a thermal desorption unit (TDU) equipped with a MPS 2 autosampler and a Peltier-cooled CIS 4 programmed temperature vaporization (PTV) inlet (GERSTEL) installed on an Agilent 7890A gas chromatograph with a 7200 Q−TOF−MS and a SCD (Agilent Technologies, Santa Clara, CA). The GC−Q−TOF−MS/SCD configuration was equipped with an Agilent capillary flow technology (CFT) three-way splitter (with makeup gas line), which was controlled with a pressure control module (PCM). The stir bars were thermally desorbed by programming the TDU from 30 °C (held for 0.5 min) to 200 °C (held for 3 min) at 720 °C/min with 50 mL/min desorption flow. Desorbed compounds were focused at 10 °C on a liner packed with quartz wool in the Peltier-cooled PTV inlet for subsequent GC−Q−TOF−MS/SCD analysis. After desorption, the PTV inlet was programmed from 10 to 240 °C (held for GC run time) at 720 °C/min to inject trapped compounds onto the analytical column. The injection was performed in the splitless mode, and the split valve was closed for 3 min. Separations were performed on a 30 m × 0.25 mm inner diameter, 0.25 μm film thickness DB-Wax column (Agilent). The column temperature for the DB-Wax was programmed from 40 °C (held for 3 min) to 250 °C (held for 20 min) at 10 °C/min. After the retention time of 33 min, the capillary column was back-flushed. The inlet pressure was 362 kPa, and the pressure of the PCM for the three-way splitter was 25 kPa. The constant pressure mode was used with helium carrier gas. A deactivated fused silica capillary with 1.1 m × 0.25 mm inner diameter was used for connecting from the splitter to the SCD, and a deactivated fused silica capillary with 1.1 m × 0.20 mm inner diameter was used for connecting from the splitter to the Q−TOF− MS. These pressures and transfer-line capillaries allow for parallel MS and SCD with minimum delay time (typically less than 0.1 s) at a constant split ratio of 1:2. The Q−TOF−MS was operated at a mass range of m/z 29−500 with dual gain mode {resolution was approximately 7000 full width at half maximum [(fwhm)]}. The data acquisition speed was 5 Hz. The electron accelerating voltage of the electron ionization (EI) was 70 eV. Methane was used as a reagent gas at 1.0 mL/min in the positive chemical ionization (PCI) mode. Nitrogen was used as collision gas at 1.5 mL/min, and a collision energy of 2.5−10 V was used for MS/MS

7.5 6.4 2.0 20 13 7.4 4.4 5.2 −10

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DOI: 10.1021/acs.jafc.5b02298 J. Agric. Food Chem. 2015, 63, 6698−6706

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Journal of Agricultural and Food Chemistry

Figure 4. Extraction time profiles for cis-ETP derivatives of thiols in spiked beer by der−SBSE. The peak area for 3SHol is multiplied by 30.

Figure 2. Comparison of the normalized responses of cis-ETP derivatives of thiols in spiked beer as a function of the ETP concentrations (5−50 mM).

Figure 5. Comparison of the normalized responses of cis-ETP derivatives of thiols in spiked hop as a function of the Tris−HCl concentrations (0, 5, 10, and 20 mM). Figure 3. Comparison of the normalized responses of cis-ETP derivatives of thiols in spiked beer as a function of the NaCl concentrations (0, 15, and 30%).

PCI mode. Nitrogen was used as collision gas at 1.5 mL/min, and a collision energy of 2.5−10 V was used for MS/MS experiments. Data Analysis. MassHunter qualitative analysis version B.06.00633 (Agilent) and MassHunter Quantitative Analysis version B.06.00388 (Agilent) were used for data analysis. The log Kow values are calculated with a SRC-KOWWIN version 1.68 software package (Syracuse Research, Syracuse, NY).

experiments. Alternate MS mode was used in the MS/MS measurement for simultaneous TOF−MS and Q−TOF−MS (MS/MS) detection. TOF−MS calibration was performed with perfluorotributylamine (PFTBA) for EI and perfluoro-5,8-dimethyl-3,6,9-decane trioxide (PFDTD) for PCI, respectively, in every sequence. Thermal Desorption−Gas Chromatography−Triple-Quadrupole Mass Spectrometry (TD−GC−QQQ−MS). For the optimization of the method parameters and the determination of the ETP derivatives of thiols, TD−GC−MS/MS in the SRM mode using the GERSTEL MPS2/TDU/CIS system installed on the Agilent 7890GC combined with a 7000B triple-quadrupole (QQQ) MS was used. GC−QQQ−MS was equipped with the PCM and a purge tee option for back-flush capability. The same TD condition described in the previous section but with the liquid nitrogen cooling for the PTV inlet (because of the higher GC initial temperature of 100 °C) was used for the TD−GC−QQQ− MS analysis. Separations were performed on a 15 m × 0.25 mm inner diameter, 0.25 μm film thickness DB-Wax column (Agilent). A 0.75 m × 0.15 mm inner diameter uncoated deactivated postcolumn (Agilent) was used and connected between the purge tee and the QQQ−MS. Column flow was set at a constant helium flow of 1.2 mL/min. The column temperature for the DB-Wax was programmed from 100 °C (held for 3 min) to 250 °C (held for 11 min) at 10 °C/min. After the retention time of 19 min, the capillary column was back-flushed. The QQQ−MS was operated in three acquisition modes: (1) scan mode at a mass range of m/z 29−500, (2) product ion scan mode at a mass range of m/z 29−500, and (3) SRM mode with the selected transitions (precursor to product ion). Transitions of the analytes are listed in Tables 2 and 3. The electron accelerating voltage of the EI was 70 eV. Methane was used as a reagent gas at 1.0 mL/min in the

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RESULTS AND DISCUSSION Identification of ETP Derivatives of Thiols. Because reference compounds of the ETP derivatives of the target thiols are not commercially available, identification of the ETP derivatives was first investigated. der−SBSE of spiked water at 10 ng/mL (adjusted at pH 9 and containing 5% ethanol and 10 mM ETP) was performed for 60 min with the PDMS stir bar. After extraction, the PDMS stir bar was analyzed with TD−GC−Q−TOF−MS/SCD. The peaks in the SCD chromatogram clearly indicate the presence of sulfur compounds. From the corresponding peaks in a total ion chromatogram (TIC), EI mass spectra of ETP derivatives of thiols (4S4M2Pone-ETP, 3SHol-ETP, and 3SHA-ETP) were obtained for both cis and trans stereoisomers. Previous studies22 reported that the major isomer is likely to be cis form. Although molecular ions were observed for all analytes with mass errors of less than 1.3 mDa (5.4 ppm), the relative abundances of those ions were very small (especially for 3SHol). To determine the molecular mass of these compounds, PCI was also performed. The protonated molecule (MH+) and adduct ions ([M + C2H5] + and [M + C3H5] +) from PCI with methane could be observed for all analytes, with the mass errors in the range of 0.69−2.55 mDa (2.5−9.3 ppm). Finally, structure elucidation was performed with collisioninduced dissociation (CID) [also known as collisionally activated 6701

DOI: 10.1021/acs.jafc.5b02298 J. Agric. Food Chem. 2015, 63, 6698−6706

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Journal of Agricultural and Food Chemistry

Table 2. Selectivity and Sensitivity in GC−EI−MS/MS Analysis for cis-ETP Derivatives of 4S4M2Pone, 3SHol, and 3SHA EI fragment ion precursora

M+ precursor

compound cis-4S4M2Pone−ETP cis-3SHol−ETP

cis-3SHA−ETP

precursor ionb,c

collision energy (V)

230 230 232 232

2.5 2.5 2.5 2.5

132 99 152 141

274 274

2.5 2.5

241 214

product ion

b,c

sensitivity (S/N ratio)e

precursor ionb,c

collision energy (V)

○ ○ ○ ○

64 28 18 33

○ ○

27 41

195 195 214 214 214 241 241 241

5.0 5.0 5.0 5.0 5.0 5.0 5.0 5.0

selectivity

d

sensitivity product ionb,c selectivityd (S/N ratio)e 195 167 185 141 127 181 135 107

× × × × ○ × × △

n/a n/a n/a n/a 29 n/a n/a 18

a

The highest intensity ion as well as having higher m/z than that of the molecular ion of the non-derivative form. bThe ions shown in bold were used for SRM transitions. cThe ions shown in underline were used for quantification. dSelectivity was shown as follows: ○, targeted peak (cis form) was separated from interferences; △, targeted peak was co-eluted with interferences, but the targeted peak in the spiked beer was clearly higher than those in the non-spiked beer; and ×, targeted peak was co-eluted with interferences. eSensitivity was shown by the signal-to-noise ratio of the targeted peak in the spiked beer.

dissociation (CAD)23] of the protonated molecule (MH+ ± 0.5 mDa). It is known that fragmentation of the protonated molecule generally occurs in the protonated part (function) of a compound,24,25 resulting in a simple bond cleavage process compared to the more extensive EI fragmentation. Therefore, CAD of the protonated molecule in accurate mass measurement can significantly help structure elucidation. Figure 1 demonstrates the CAD mass spectra of the protonated molecule of 4S4M2Pone−ETP using the collision energy of 2.5 V. Also, structures and bond cleavages of the analytes are shown in Figure 1. Considerable information concerning the structures of ETP derivatives of thiols can be derived from the fragment ions in the CAD mass spectra. For 4S4M2Pone−ETP [CH3CH2O(CO)CHCHSC(CH3)2CH2(CO)CH3 + H]+], the mass error of the protonated molecule (m/z 231.1003) was 0.46 mDa (2.0 ppm). The most abundant ion at m/z 185.0619 corresponds to the bond cleavage 1 and [MH − CH3CH2OH]+, with a mass error of 1.2 mDa/6.4 ppm, and the ion at m/z 99.0797 corresponds to the bond cleavage 2 and [MH − CH3CH2O(CO)CHCHSH]+, with a mass error of 0.74 mDa/7.5 ppm. The neutral loss of CH3CH2O(CO)CHCHSH demonstrates the derivatization of the mercapto function of thiols with ETP (CH3CH2O(CO)CHCH). These ions clearly demonstrate that the structures, such as CH3CH2O− and CH3CH2O(CO)CHCHS−, are involved. The neutral loss of CH3CH2O(CO)CHCHSH and CH3CH2OH is also observed for 3SHol−ETP (m/z 101.0941, mass error of 2.0 mDa/20 ppm; m/z 187.0762, mass error of 2.4 mDa/13 ppm) and 3SHA−ETP (m/z 143.1056, mass error of 1.1 mDa/7.4 ppm; m/z 229.0881, mass error of 1.2 mDa/ 5.2 ppm). Consequently, the ETP derivatives of three thiols could be identified with molecular mass determination, formula calculation, and structure elucidation. The mass spectral information used for the identification of the ETP derivatives is summarized in Table 1. Optimization of der−SBSE Conditions for the Beer Sample. To optimize der−SBSE of thiols with ETP in beer matrices, the influence of varying concentration of ETP was first examined with the spiked beer. The pH of the spiked beer was adjusted to 9, and the spiked concentration of thiols was 10 ng/mL each. The concentration of ETP was varied with the addition of a 50% ethanol solution of ETP. SBSE with 60 min extraction was performed in duplicate for 10 concentrations of

ETP between 5 and 50 mM. Figure 2 demonstrates a comparison of normalized responses of the cis-ETP derivatives as a function of ETP concentrations. For 3SHA−ETP, which has the highest log Kow of 3.45 (the most hydrophobic of the analytes), the ETP concentrations of 20 mM showed the largest response and the ETP concentrations of 10 and 15 mM also showed a similar large response (94 and 99% of the largest response, respectively). However, the responses decreased with ETP concentrations of more than 25 mM. For 4S4M2Pone− ETP (log Kow of 1.83) and 3SHol−ETP (log Kow of 2.45), the normalized responses gradually increased with increasing ETP concentrations from 5 to 35 mM and the ETP concentration of 35 mM showed the largest response. However, the responses dramatically decreased with the ETP concentrations of more than 40 mM. It was observed that the ETP concentrations of more than 40 mM induced a visible “droplet” of ETP in beer matrices. This “oil effect” might influence the reaction and/or the kinetics between thiols, ETP, ETP derivatives (thioacrylates), beer matrices, and the PDMS coating of the stir bar in der− SBSE, even before the appearance of the visible droplet. This relationship is quite complex, but it is clear that the solubility of ETP in sample matrices is a critical parameter. This parameter should be considered when the method is applied for nonalcoholic sample matrices, such as hop water extract and wort, which might have low ETP solubility without percent levels of ethanol. Because 4S4M2Pone has the lowest odor threshold level in beer at 1.5 ng/L4 compared to those of 3SHol at 55 ng/L4 and 3SHA at 5.0 ng/L,5 the parameters should be optimized for the condition that gives the best sensitivity of 4S4M2Pone. Therefore, the ETP concentration of 35 mM was selected for further study. Previous studies have shown that salt addition increases the recovery of polar solutes with low log Kow values (e.g., log Kow < 2.5).19,20 Therefore, the effect of salt addition was examined with the spiked beer at 10 ng/mL containing 35 mM ETP. der−SBSE was performed for 50 min in duplicate. Figure 3 demonstrates a comparison of normalized responses of the cisETP derivatives as a function of the NaCl concentrations (0, 15, and 30%). As expected, the responses for 4S4M2Pone−ETP (log Kow of 1.83) and 3SHol−ETP (log Kow of 2.45) significantly increased with the salt addition and the 30% salt addition gives the largest responses. However, 3SHA−ETP (log Kow of 3.45) 6702

DOI: 10.1021/acs.jafc.5b02298 J. Agric. Food Chem. 2015, 63, 6698−6706

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6703

The highest intensity ion as well as having higher m/z than that of the molecular ion of the non-derivative form. bThe ions shown in bold were used for SRM transitions. cThe ions shown in underline were used for quantification. dSelectivity was shown as follows: ○, targeted peak (cis form) was separated from interferences; △, targeted peak was co-eluted with interferences, but the targeted peak in the spiked beer was clearly higher than those in the non-spiked beer; and ×, targeted peak was co-eluted with interferences. eSensitivity was shown by the signal-to-noise ratio of the targeted peak in the spiked beer.

cis-3SHA−ETP

cis-3SHol−ETP

a

n/a 22 n/a n/a 53 55 87 × ○ × × ○ ○ ○ 187 103 83 229 187 143 83 2.5 2.5 2.5 2.5 2.5 2.5 2.5 n/a 0.80 n/a n/a 3.6 261 261 261 303 303

5.0 5.0 5.0 5.0 5.0

261 187 115 303 215

× △ × × △

187 187 187 229 229 229 229

n/a 11 49 × △ △ 185 99 87 2.5 2.5 2.5 185 185 185 n/a n/a n/a × × × 259 161 133 5.0 5.0 5.0 259 259 259

n/a n/a 3.7 0.60 n/a n/a n/a n/a n/a n/a × × △ △ × × × × × × 231 185 133 99 233 187 135 229 187 143 2.5 2.5 2.5 2.5 2.5 2.5 2.5 5.0 5.0 5.0 231 231 231 231 233 233 233 275 275 275

product ionb,c selectivityd product ionb,c selectivityd compound

cis-4S4M2Pone−ETP

collision energy (V) precursor ionb,c sensitivity (S/N ratio)e collision energy (V) precursor ionb,c sensitivity (S/N ratio)e collision energy (V) precursor ionb,c

PCI

[M + C2H5]+ precursor MH+ precursor

Table 3. Selectivity and Sensitivity in GC−PCI−MS/MS Analysis for cis-ETP Derivatives of 4S4M2Pone, 3SHol, and 3SHA

fragment ion precursora

shows the largest response with the 15% salt addition, and the 30% salt addition results in nearly the same response as with the non-salt addition. It is known that the salt addition decreases the recovery of more hydrophobic solutes (e.g., log Kow > 4.0) in SBSE.19,20 Although the log Kow value of 3SHA−ETP is lower than 4.0, the oil effect of ETP might be promoted with the 30% salt addition, resulting in the reduced response of 3SHA−ETP (in comparison to the 15% salt addition). To obtain the best sensitivity for 4S4M2Pone, which has the lowest odor threshold level, the 30% salt addition was selected for further study. Finally, the extraction time profile was examined with five extraction times between 30 and 240 min. Duplicate analyses were performed for each extraction time. Figure 4 shows the extraction time profiles of the cis-ETP derivatives in the spiked beer. Equilibrium was nearly reached at 120 min for 3SHol−ETP and 180 min for 4S4M2Pone−ETP and 3SHA−ETP. Although it is necessary to have 180 min extraction for the maximum recovery for 4S4M2Pone (and 3SHA), the der−SBSE method can be performed in parallel (up to 20 samples on one stir plate) and does not require any additional manual steps after extraction (before TD−GC analysis). Therefore, the extraction time of 180 min was selected for further study. Optimization of der−SBSE Conditions for Hop Water Extracts and Wort Samples. The optimized der−SBSE condition with the spiked beer in the previous section was applied to the spiked beer, hop water extract, and wort at 1 ng/mL (except for 3SHol at 10 ng/mL) in duplicate. All samples were adjusted to pH 9 using NaOH solution (1 M). In comparison to the spiked beer, ETP derivatives of all target thiols were, however, much more weakly detected in the hop water extract and were not detected in the wort. This is due to the low ETP solubility of both hop water extract and wort, which do not contain percent level ethanol, resulting in the “oil effect” (visible “droplet”) of ETP in the samples. To give a similar ETP solubility with beer, hop water extract and wort were 2-fold diluted with 10% ethanol solution (resulting in 5% ethanol in the diluted sample). Then, the same der−SBSE conditions were applied to the spiked hop water extract and wort again. For the spiked wort, ETP derivatives of all target thiols were detected with nearly the same abundance as the spiked beer. However, for the spiked hop water extract, 4S4M2Pone− ETP and 3SHol−ETP were still lower than those of the spiked beer and wort. This might be due to lack of buffer capacity of hop water extract, which leads to changes in pH during the derivatization. It is known that both beer and wort have relatively similar buffer capacities that resist changes in pH on the addition of acids or bases.25 To examine the effect of buffer in the hop extract, Tris−HCl (1 M, pH 9) was added to the spiked hop water extract. Figure 5 shows a comparison of normalized responses of cis-ETP derivatives as a function of the Tris−HCl concentrations (0, 5, 10, and 20 mM). The response for 4S4M2Pone−ETP notably increased with the buffer addition, and 5 mM gives the largest response. The response for 3SHol− ETP also increased with the buffer addition, and similar responses are seen with all buffered conditions. However, the response for 3SHA−ETP decreased with the buffer addition, especially for 10 and 20 mM. Therefore, the 5 mM Tris−HCl addition was selected for the hop extract sample. Selectivity and Sensitivity in GC−MS/MS Analysis. To evaluate the selectivity and sensitivity of the method, the spiked water at 1−10 ng/mL and non-spiked and spiked beer at 10−100 pg/mL were analyzed with several GC−MS/MS conditions, including different ionization modes (EI and PCI), collision energies, and SRM transitions. We selected the following

sensitivity product ionb,c selectivityd (S/N ratio)e

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Journal of Agricultural and Food Chemistry Table 4. Linearity, LOD, and Apparent Recovery of cis-ETP Derivatives of 4S4M2Pone, 3SHol, and 3SHA SRM transitions

apparent recovery

compound

target

qualification

r2

LOD (ng/L)

recovery (%)

RSD (%) (n = 6)

cis-4S4M2Pone−ETP cis-3SHol−ETP cis-3SHA−ETP

230 > 132 232 > 141 274 > 214

230 > 99 232 > 152, 214 > 127 274 > 241

0.9999 0.9976 0.9943

0.20 27 0.19

99 101 101

2.8 1.3 7.2

Figure 6. SRM chromatograms obtained by der−SBSE−TD−GC−QQQ−MS of spiked and non-spiked beer hopped with Citra hop: (1′) trans4S4M2Pone−ETP, (1) cis-4S4M2Pone−ETP, (2′) trans-3SHA−ETP, (2) cis-3SHA−ETP, (3′) trans-3SHol−ETP, and (3) cis-3SHol−ETP. Spiked concentrations are 40 ng/L for 4S4M2Pone and 3SHA and 400 ng/L for 3SHol.

that involves a calibration graph26) of the method. The standard addition calibration method and the spiked beer sample were used for the evaluation. The peak area of the selected ion from the cis-ETP derivative was used for both calibration and determination. Linearity was assessed at six concentration levels between 1 and 100 ng/L for 4S4M2Pone and 3SHA in the beer sample, which contains these compounds at non-detectable levels (below sub-nanogram per liter) with the proposed method. Because detectable levels of 3SHol (>20 ng/L) are present in most beer samples, the linearity between 20 and 1000 ng/L for 3SHol was evaluated. For each level, duplicate analyses were performed. For all compounds, good linearity was achieved with r2 above 0.9943. The LOD for 4S4M2Pone and 3SHA was estimated using low-concentration spikes (at 1 ng/L) in the same beer samples used for the linearity study and calculating the standard deviation of the determination. The LOD is then defined as 3 times the standard deviation (for six replicates) obtained for the analyte concentration not greater than 10 times the LOD.27 The LOD for 3SHol was estimated using the blank determination procedure28 because of the presence of 3SHol at the detectable levels in the beer samples. The LOD for 3SHol is then expressed as the analyte concentration corresponding to the sample blank value plus 3 times the standard deviation.28 For all analytes, the LODs were below their odor threshold levels in the range of 0.19−27 ng/L. The apparent recovery was assessed by six replicate analyses of the spiked beer samples. The spiked levels were 10 ng/L for 4S4M2Pone and 3SHA and 100 ng/L for 3SHol. The apparent recoveries were good for all analytes in the range of 99−101% [relative standard deviation (RSD) of 1.3−7.2%; n = 6]. The linearity, LOD, and apparent recovery of the method are summarized in Table 4. Analysis of Beer, Hop Water Extract, and Wort Samples. To demonstrate the applicability of the method, quantitation of the targeted thiols was performed for three kinds of beer hopped with Cascade, Citra, and Nelson Sauvin hop cultivars.

precursor ions for SRM: (1) molecular ion, (2) fragment ion that shows the highest intensity as well as having higher m/z than that of the molecular ion of the non-derivative form (e.g., m/z > 132 for 4S4M2Pone), (3) protonated molecule (PCI), and (4) adduct ion (PCI). Product ion scan with EI and PCI for the precursor ions was first performed with collision energies of 2.5, 5, 10, and 20 V for the spiked water, and the collision energies that give the largest sum of the ion intensities were selected for SRM of each precursor ion. SRM with EI and PCI was then performed with the selected collision energies for non-spiked and spiked beer. The selectivity of SRM conditions was evaluated with the following criteria: (1) the targeted peaks (cis form) should be separated from the interferences, and (2) the targeted peaks in the spiked beer should be clearly higher than those in the nonspiked beer (if detected). The sensitivity of SRM conditions was evaluated with a signal-to-noise (S/N) ratio of the targeted thiols in the spiked beer. More than two acceptable transitions could be obtained for all targeted thiols, with SRM using the molecular ion or fragment ion as the precursor ion in the EI. However, no acceptable transitions and only one transition were obtained for 4S4M2Pone−ETP and 3SHol−ETP, respectively, with all SRM conditions in the PCI. Therefore, SRM transitions that give the highest S/N ratio in the EI mode were selected for quantitation. Additionally one or two SRM transitions in the EI were selected as qualifiers. The relative ratios of transition intensities (e.g., 230 > 132/230 > 99 for 4S4M2Pone−ETP) were compared to those in the spiked water, and those were less than 20%. Finally, the selection of SRM transitions for the internal standards (d10-4S4M2Pone and d2-3SHol) was performed with the same procedure but with the EI only. Tables 2 and 3 summarize the parameters used for the selectivity and sensitivity in GC−MS/MS analysis. Analytical Performance. We evaluated linearity, limit of detection (LOD), and apparent recovery (the quantity of observed value/reference value, obtained using an analytical procedure 6704

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Figure 7. Determined values obtained by der−SBSE−TD−GC−QQQ−MS of beer samples hopped with Cascade, Citra, and Nelson Sauvin hop cultivars and the corresponding hop water extracts and wort sample.

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ACKNOWLEDGMENTS The authors thank Kevin MacNamara (Dublin, Ireland) and Shigeki Daishima of Agilent Technologies Japan, Ltd. for their kind support.

The corresponding hop water extract and (hopped) wort were also analyzed. Triplicate analyses with a three-point standard addition calibration method using internal standards (d104S4M2Pone for 4S4M2Pone and 3SHA and d2-3SHol for 3SHol) were performed for each sample. The peak area of the selected ion from the cis-ETP derivative was used for both calibration and determination. Figure 6 illustrates the sum of SRM chromatograms obtained from beer hopped with Citra hop cultivar. The determined values are shown in Figure 7. For 4S4M2Pone, Citra hop and Nelson Sauvin hop show higher values (9.3−12 ng/L; RSD of 3.0−5.2%; n = 3) than that of Cascade hop (2.1 ng/L; RSD of 4.1%; n = 3). The corresponding hopped wort and beer also show a similar trend, and the increased values of all beer samples (6.3−22 ng/L; RSD of 5.1−8.2%; n = 3) indicate the formation of 4S4M2Pone during fermentation from precursors.6 Although 3SHol was not detected in all hop water extract samples, the determined values of 3SHol in hopped wort and beer are in the range of 55−93 ng/L (RSD of 11−12%; n = 3) and 180−290 ng/L (RSD of 7.5−12%; n = 3), respectively. Again, the increased values of all beer samples illustrate the effect of precursors during fermentation. As a previous study reported,10 the determined values of 3SHol between hopped wort and beer show a linear correlation with r2 of 0.9477. 3SHA was not detected in hop water extract and hopped wort and was only detected in beer (3.2−10 ng/L; RSD of 4.6−9.8%; n = 3). It is known that some yeasts have high capability of converting 3SHol into 3SHA during fermentation.29,30 However, the order of the determined values of 3SHA in beer (Nelson Sauvin > Cascade > Citra) did not reflect those of 3SHol in hopped wort and beer (Cascade > Citra > Nelson Sauvin).

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ABBREVIATIONS USED

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REFERENCES

3SHol, 3-sulfanylhexan-1-ol; 3SHA, 3-sulfanylhexyl acetate; 4S4M2Pone, 4-sulfanyl-4-methylpentan-2-one; CAD, collisionally activated dissociation; ETP, ethyl propiolate; LOD, limit of detection; PDMS, polydimethylsiloxane; Q−TOF−MS, quadrupole time-of-flight mass spectrometer; RSD, relative standard deviation; SRM, selected reaction monitoring; der−SBSE, stir bar sorptive extraction with in situ derivatization; SCD, sulfur chemiluminescence detector; MS/MS, tandem mass spectrometry; TD, thermal desorption; QQQ−MS, triple-quadrupole mass spectrometer

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AUTHOR INFORMATION

Corresponding Author

*Telephone: +81-3-5731-5321. Fax: +81-3-5731-5322. E-mail: [email protected]. Notes

The authors declare no competing financial interest. 6705

DOI: 10.1021/acs.jafc.5b02298 J. Agric. Food Chem. 2015, 63, 6698−6706

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