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Analysis of hop acids by thin-layer chromatography and the Molecular Ionization Desorption Analysis Source (MIDAS) for mass spectrometry
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Analysis of hop acids by thin-layer chromatography and the Molecular Ionization Desorption Analysis Source (MIDAS) for mass spectrometry Gregory T. Winter, Joshua A. Wilhide, William R. LaCourse ∗ University of Maryland, Baltimore County, 1000 Hilltop Circle, Baltimore, MD, 21250, USA
a r t i c l e
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Article history: Received 14 July 2017 Received in revised form 21 August 2017 Accepted 22 August 2017 Available online xxx Keywords: Ambient ionization Hop acids MIDAS Thin-layer chromatography
a b s t r a c t A thin-layer chromatography ambient mass spectrometry method was developed for the quantitative analysis of hop acids. Hop acids were extracted from three varieties of hop pellets, Apollo, Cluster and Czech Saaz. Reversed-phase thin-layer chromatography provided the optimum stationary phase for separation and ionization. Using standards, a method was developed to separate ␣-, iso-␣- and -acids by thin-layer chromatography. Each thin-layer chromatography plate was analyzed directly by the Molecular Ionization Desorption Analysis Source (MIDAS). MIDAS is an atmospheric pressure ambient ionization source for mass spectrometry. Quantitative analysis of the hop extracts was made by successive analysis of known amounts of standards and the determination of a response factor. The expected trend of total ␣-acid content with Apollo > Cluster > Czech Saaz was observed. Variation in the results was attributed to manual TLC plate spotting and not to the ionization source. © 2017 Elsevier B.V. All rights reserved.
1. Introduction Hops are the flowers of the female variety of the plant Humulus lupulus [1]. Hops are used in the beer brewing process to add bitterness and aroma to the final product. While there are a variety of compounds that contribute to the aromatic flavors in beer, there are a few primary compounds that contribute bitterness. These compounds, known as alpha acids (␣-acids), are humulone, adhumulone and cohumulone. In their native form, however, the ␣-acid compounds are not readily soluble in water and do not contribute any bitter flavor [2]. When hops are added during the boiling process used in making beer, the ␣-acids undergo thermal isomerization, via an acyloin-type ring contraction, to the more water soluble, bitter tasting, iso-␣-acids [2]. Also found in hops are three primary beta acids (-acids) including lupulone, adlupulone and colupulone. The oxidation products of these acids, such as hulupinic acid, can contribute additional bitterness to the final product [1]. Determining the content of ␣- and -acids in a sample of hops, whether they be whole leaf, pellet or extract is of primary importance in the brewing industry. Many validated methods exist for this purpose, primarily using High Performance Liquid Chromatography (HPLC) with UV–vis detection [3–6]. In addition to these validated methods, some researchers have developed their own [7–9].
In this paper, thin-layer chromatography (TLC) is used to separate ␣- and -acids extracted from hop pellets. TLC offers the advantage of being simple and cost effective, using very little solvent. TLC has been used for the separation of ␣- and -acids previously [10–12]. Typically, in order to identify a spot on a TLC plate, the plate either needs to be stained with a particular reagent or the spot removed and analyzed in a separate experiment. Presented here is the direct analysis of ␣- and -acids extracted from hop pellets from the surface of a TLC plate using the Molecular Ionization Desorption Analysis Source (MIDAS). MIDAS is a recently introduced ambient ionization and sampling platform for mass spectrometry [13]. Previous characterization of beer and hops has employed ambient ionization, including atmospheric pressure chemical ionization (APCI) [14] and direct analysis in real time (DART) [15,16]. For comparison and to establish a reference value for the acid content in each hop sample, ASBC method HOP-6A was used [17]. This method is a UV–vis spectrophotometric method for determining total ␣- and -acid content in hop samples. The goal of these experiments is to assess the potential for using TLC and MIDAS for quantitative determination of ␣- and -acids from hop pellets.
2. Materials and methods 2.1. Standards and materials
∗ Corresponding author. E-mail address: [email protected] (W.R. LaCourse).
Methanol, toluene, sodium hydroxide, formic acid and phosphoric acid (85%) were purchased from Fisher Scientific (Pittsburgh,
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Fig. 1. Structures of ␣, iso-␣, and  acids.
PA). C18 modified silica gel 60 plates, 5.0 × 10.0 cm, 250 m thickness, (EMD Millipore, Billerica, MA) were used for ␣-, iso-␣- and -acid separation. Additional testing was done on normal-phase silica gel 60 plates, 2.5 × 7.5 cm, 250 m thickness (EMD Millipore, Billerica, MA). Certified standards, International Calibration Standard I3 (ICSI3) and International Calibration Extract 3 (ICE-3) were purchased from the American Society of Brewing Chemists (St. Paul, MN). ICS-I3 contains the trans form of the dicyclohexylamine complex (DCHA)-iso-␣-acid of humulone, adhumulone and cohumulone (62.3% w/w). ICE-3 contains the un-isomerized forms of humulone, adhumulone and cohumulone (44.64% w/w), as well as the -acids lupulone, adlupulone, and colupulone (24.28% w/w). Structures of these compounds can be found in Fig. 1. Pelletized hops of the Apollo, Cluster, and Czech Saaz variety were purchased from a local homebrew supply store. These varieties were chosen as they span the low, medium and high ␣-acid content range, respectively. As stated on the packaging, the Apollo, Cluster and Czech Saaz varieties contained 17%, 8.1% and 2.6% ␣-acid with -acid content of 6.7%, 5.2% and 4.7%, respectively. 2.2. Standards preparation To prepare the ICE-3 standard, 550.8 mg was placed in 20.00 mL of methanol and sonicated until dissolved. This solution was then diluted to a final volume of 100.00 mL with methanol. Based on the certified concentration, this dilution results in 0.597 mg mL−1 of lupulone and adlupulone, 0.740 mg mL−1 of colupulone, 1.694 mg mL−1 humulone and adhumulone and 0.765 mg mL−1 of cohumulone. ICS-I3 was dissolved to a concentration of 1.300 mg mL−1 in methanol. 2.3. Hop extract preparation Hop extracts prepared using this method were used for both the UV and TLC experiments. Hop pellets were ground until the entire sample could be passed through a number 20 stainless steel sizing screen. 50.00 mL of toluene was then added to 2.5000 (±0.0015) g of ground hop pellets in an Erlenmeyer flask. The flask was cov-
ered in foil to protect against light and shaken for 30 min. After shaking, the sample was centrifuged at 1800 rpm for five minutes. The supernatant was then collected for analysis. All samples were stored at −20 ◦ C under a nitrogen atmosphere until analysis. These compounds are known to be light, heat and oxygen sensitive and care should be taken to minimize their exposure to the atmosphere during preparation and storage. 2.4. UV method To prepare the blank, 5.00 mL of toluene was added to a 100.00 mL volumetric flask and brought to volume with methanol, creating dilution A. To prepare dilution B, 3.00 mL of dilution A was added to a 50.00 mL volumetric flask and brought to volume with alkaline methanol. To prepare alkaline methanol, 0.20 mL of 6.0 N sodium hydroxide was added to 100.00 mL of methanol. Each hop extract sample was prepared in the same manner as the blank, with the initial 5.00 mL being the hop extract. After preparation, samples were run immediately on a JASCO V560 spectrophotometer (Easton, MD). The instrument was run in scanning mode from 270 to 450 nm. Each sample was analyzed five times with fresh sample placed in the cuvette for each analysis. 2.5. Thin-layer chromatography-MIDAS method For method development, four L of a 1:1 mixture of ICS-I3 and ICE-3 was spotted onto the TLC plates. Plates were developed in either 100% methanol; 1% phosphoric acid in methanol (v/v); or 1% formic acid in methanol (v/v). For quantitative analysis of both the standards and sample, 1.0 L of each was manually spotted onto a TLC plate using a glass microsyringe. The plate was then developed in a 1% solution of formic acid in methanol (v/v). Due to the high concentration of ␣-acids in the Apollo and Cluster samples, these solutions were further diluted 1:1 (v/v) in methanol prior to analysis. 2.6. Mass spectrometer Mass spectrometer tuning parameters were the same as those used previously [1]. Briefly, analyses were performed on
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Table 1 ␣ and -acid content by technique including original package values. Hop Variety
Technique
(w/w%) ␣-acid content (±95% CI)
(w/w%) -acid content (±95% CI)
Ratio Total ␣: acid content (%RSD)
Apollo
UV–vis TLC-MIDAS Package
18.17 (0.12) 17.7 (7.1) 17
4.85 (0.10) 6.2 (1.9) 6.7
3.75 (2.20) 2.66 (6.89)a 2.5
Cluster
UV–vis TLC-MIDAS Package
7.31 (0.05) 3.7 (0.9) 8.1
4.18 (0.04) 3.8 (0.7) 5.2
1.75 (1.39) 0.88 (20.10)a 1.6
Czech Saaz
UV–vis TLC-MIDAS Package
2.84 (0.03) 1.9 (0.5) 2.6
4.44 (0.03) 7.4 (1.3) 4.7
0.64 (1.46) 0.22 (2.65)a 0.6
a
Ratios were determined from an average of absolute peak areas, not from values determined by response factors.
(w/w%)˛-acidcontent = d × (−51.56A355 + 73.79A325 − 19.07A275 )
(2)
(w/w%)ˇ-acidcontent = d × (−55.57A355 + 47.59A325 + 5.10A275 )
(3)
Table 1 contains the average ␣- and -acid content calculated for each of the three hop varieties with the associated 95% confidence interval (CI). 3.2. Hop acid separation and MIDAS method development
Fig. 2. UV absorbance spectra for Apollo, Cluster and Czech Saaz hop varieties.
a PerkinElmer AxION Time-of-Flight mass spectrometer operated in negative ion mode. Negative mode parameters include capillary exit, skimmer, RF and offset voltages of −100 V, −20 V, −450 V and −10.3 V, respectively. Spectra were acquired at a rate of 2 spectra s−1 . 2.7. MIDAS parameters Nitrogen gas temperature was maintained at 180 ◦ C with a supply pressure of 40 psi and a purity of >99%. The corona discharge was operated with a negative polarity at −2.5 kV and 12 A. Atmospheric relative humidity ranged between 18 and 50%. No additional reagents or solvents were added to the system. For all analyses, the TLC plate was moved through the sampling region at a rate of 0.3 mm s−1 . 3. Results and discussion 3.1. UV experiment Each hop sample produced a similar UV spectrum with the absorbance intensity increasing with greater ␣- and -acid content. Fig. 2 contains an example UV absorbance spectrum for each of the three hop varieties tested. The absorbance maxima at the wavelengths of 355, 325 and 275 nm are used to calculate the ␣ and  acid content in each sample. There are three equations contained within the method. Eq. (1) accounts for the dilution of the sample, Eq. (2) calculates the ␣-acid content, and Eq. (3) calculates the -acid content. d=
vol.dil.A (mL) × voldil.B (mL) 500 ∗ aliq.extract (mL) × aliq.dil.A (mL)
(1)
To ensure that, if present, any iso-␣-acids would be detected, it was important that the TLC method successfully separate the iso-␣acids from the ␣-acids, as the acid isomers have the same molecular weight and cannot be distinguished by mass spectrometry alone. Separating the iso-␣-acids from the ␣-acids may also allow this method to be used for finished beer. Historically, TLC separations involving ␣- and -acids used normal-phase plates [10–12]. For this application, however, no ionization was achieved when testing these compounds on normal-phase plates. The lack of ionization is likely due to significant non-covalent bonding of the analyte molecules to the stationary phase of the TLC plate. Therefore, reversed-phase TLC plates were used. These plates also have the advantage of employing solvent conditions similar to those used in HPLC experiments, making method development more straightforward. Initially, a 1% solution of phosphoric acid in methanol (v/v) was used as the mobile phase. Phosphoric acid was chosen as it is a common modifier used in HPLC separations of these compounds [8]. It was not expected that a modifier would adversely impact ionization or detection because there was no direct introduction of the mobile phase to the instrument, unlike in traditional HPLC techniques. Under these solvent conditions, three distinct groups of compounds corresponding to the ␣-, iso-␣- and -acids were found when observing the TLC plate under a UV lamp. Complete separation of each subset of compounds is not required as each mass can be extracted individually from the total ions observed, providing sufficient information to determine the total amount of each type of compound present. While some ionization of each acid was observed, signal intensity was inconsistent and not reproducible. The likely cause of the inconsistent ionization was the phosphoric acid used in the mobile phase. Phosphoric acid remained on the TLC plate after the plate had dried. The result of the remaining phosphoric acid was the generation of phosphoric acid clusters when the plate was scanned using the MIDAS. With successive scans of the TLC plates, the intensity of the phosphoric acid clusters continued to increase, indicating that carryover is a problem when using this method. The increase in ion background resulted in suppression of the analyte signal. Therefore, phosphoric acid was removed and the plate developed using 100% methanol as the mobile phase. With this solvent, however, the ␣and iso-␣-acids failed to separate. An extracted ion chromatogram (EIC) of this experiment can be found in Fig. 3A.
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Fig. 3. (A) Extracted ion chromatogram of a mixture of ICE-3 and ICS-I3 developed using 100% methanol (B) extracted ion chromtatogram of a mixture of ICE-3 and ICS-I3 developed in a 1% solution of formic acid in methanol (v/v).
Fig. 4. Example mass spectra for: (A) humulone, adhumulone, isohumulone and isoadhumulone, (B) cohumulone and isocohumulone, (C) lupulone and adlupulone, (D) colupulone.
The failure to adequately separate the compounds results from the lack of an acidic mobile phase. There is not enough difference in polarity between the ␣- and iso-␣-acids in 100% methanol to separate effectively on a 10 cm TLC plate. A 1% solution (v/v) of phosphoric acid in methanol has a pH of approximately 2 at room temperature. The pKa’s of the ␣-acids are in the range of 4.0–5.5, while the iso-␣-acids are in the range of 5.5–7.8 [18,7]. Thus, at the pH of the phosphoric acid mobile phase, the ␣-acids will be nearly
fully protonated, and therefore more non-polar, while the iso-␣acids will have a mixture of protonated and ionized species. This difference in protonation allows for the effective separation of the two groups on the TLC plate. In order to maintain a similar pH while minimizing interferences, formic acid was chosen to replace the phosphoric acid. A 1% solution (v/v) of formic acid in methanol results in a pH of 2.2 at room temperature. Formic acid is a volatile acid that is used as a
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modifier in HPLC–MS experiments. Due to its volatility, any formic acid remaining on the plate after it had dried was minimal, resulting in a cleaner background with no clusters observed. A mixture of ICE-3 and ICS-I3 was developed on a plate using a 1% solution (v/v) of formic acid in methanol. An EIC of this separation can be found in Fig. 3B. Using formic acid, the three groups of compounds were successfully separated. Example mass spectra obtained for each acid, observed as the deprotonated species [M−H]− , can be found in Fig. 4. As many of these compounds are isomeric, they are observed at the same mass to charge (m/z) ratio with the only distinguishing factor being the different retention times determined from the TLC separation.
3.3. Hop sample analysis In order to quantify the amount of each acid contained in each hop sample a response factor based on the ICE-3 standard was first determined. The response factor was determined by analyzing a known quantity of standard that had been spotted and developed on the plate. The response factor was calculated as the peak area divided by the amount of standard on the plate (in mg). The concentration in each sample was then calculated by multiplying the peak area obtained from the unknown by the response factor and any appropriate dilution factors. In an attempt to compensate for any variability between TLC plates, each plate was cut in half. Three replicates of standards were run on one half while three replicates of samples were run on the other. Each sample therefore was analyzed six times, while a total of 18 standard replicates were analyzed. The total ␣- and -acid content in each sample was calculated based on the average response factor determined from the corresponding standard run. Example EIC’s obtained for the analysis of Apollo, Cluster and Czech Saaz hop varieties are contained in Fig. 5. The calculated ␣and -acid contents with corresponding 95% confidence interval (CI) are contained in Table 1. It is worth noting that it took approximately 20–25 min to develop each plate and only four minutes to analyze each spot using MIDAS. Therefore, a single plate containing three replicates can be developed and analyzed in the time it takes to obtain a single chromatogram by conventional HPLC methods, offering savings in terms of time and solvent cost. Variation was observed in the response factors determined for the standards. When peak areas for each of the 18 acid standard trials were compared, relative standard deviation (RSD) values ranged from 38 to 51%. RSDs determined for the individual samples were also high, ranging from 16 to 35%. The tabulated results show that semi-quantitative data is being produced using this method, as the results follow the expected trend of ␣-acid content. There is, however, too much variation for the data to be considered reliable for full quantitation. Encouragingly, the variation can be attributed to the manual TLC plate spotting technique. Evidence of this was found when comparing the ratios of peak areas for total ␣: acid content. While the RSDs for total ␣- and - acid peak areas determined from the 18 replicates of the standard ICE-3 were 50.17% and 40.39% respectively; the ratio of total ␣: was found to be 1.67 with an RSD of 10.45%. The calculated ratio based on the certified values in the ICE-3 standard is 1.84. This result indicates that despite the variation in absolute peak area, the ratio remains consistent, pointing to inconsistencies in plate spotting. For the three hop samples, the ratios of total ␣: acid, as determined by total peak area (not response factor) for Apollo, Cluster and Czech Saaz are included in Table 1. Also included in Table 1 are the original package values for reference. Comparison with the package values is difficult as no certificate of analysis is available and it is unknown how the package values were obtained.
Fig. 5. EICs obtained for extracts of (A) Apollo (B) Cluster and (C) Czech Saaz hop varieties.
Deviation from the values obtained using the UV–vis method could result from bias within the method due to interfering species present in the whole hop extract. Additionally, the TLC-MIDAS still may suffer from incomplete or inconsistent ionization, but it is difficult to tell from these results given the variation in plate spotting.
4. Conclusions While this current experiment is focused on extracts from hop pellets containing ␣- and -acids, this method has been shown to successfully separate the iso-␣-acids from the un-isomerized
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forms. Therefore, this method could be extended to the analysis of isomerized hop extracts and potentially to finished beer. The goal of these experiments was to determine the feasibility of using TLC and MIDAS analysis to quantify the ␣- and -acid content in hop pellets. The accepted UV method was run in order to obtain values for the actual ␣- and -acid content in each sample, ensuring that a known value was used for comparison with the mass spectrometry results and not an assumed value from packaging, as product degradation is possible depending on storage time and conditions. The MIDAS-TLC method shows semi-quantitative results, with the determined ␣-acid content for each hop variety following the expected trend of Apollo > Cluster > Czech Saaz. While there was wide variation in the raw data, the lower RSDs found within the ratio of total ␣: content in the standards as well as the samples indicates that with further development of the TLC spotting method, fully quantitative results and an effective comparison between methods should be possible. Using a suitable internal standard may eliminate problems relating to manual plate spotting. Alternatively, the use of an automatic TLC spotter could be explored. Funding This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors. Acknowledgements The authors would like to acknowledge Ian Shaffer of the Molecular Characterization and Analysis Complex at UMBC for technical support and Maggie LaCourse for editing and review. References [1] S. Hieronymus, For the Love of Hops, vol. 178, Brewers Publications, Boulder, Colorado, 2012, pp. 19. [2] B. Jaskula-Goiris, G. Aerts, L. De Cooman, Hop ␣-acids isomerisation and utilisation: an experimental review, Cerevisia 35 (2010) 57–70. [3] ASBC Methods of Analysis, Online. Hops Method 9. ␣- and - iso-␣-Acids by High-Performance Liquid Chromatography, American Society of Brewing Chemists, St. Paul, MN, U.S.A, 2008, http://dx.doi.org/10.1094/ASBCMOAHops-9, Approved 1987, rev.
[4] ASBC Methods of Analysis, Online. Hops Method 14. ␣-Acids and - Acids in Hops and Hop Extracts by HPLC, American Society of Brewing Chemists, St. Paul, MN, U.S.A, 2008, http://dx.doi.org/10.1094/ASBCMOA-Hops-14, Approved 1990, rev. [5] ASBC Methods of Analysis, Online. Hops Method 15. Iso-␣-Acids in Isomerized Hop Pellets by HPLC, American Society of Brewing Chemists, St. Paul, MN, U.S.A, 2008, http://dx.doi.org/10.1094/ASBCMOA-Hops-15, Approved 1993, rev. [6] ASBC Methods of Analysis, Online. Hops Method 16. Iso- ␣ –, ␣-, and - Acids in Hop Extracts and Isomerized Hop Extracts by HPLC, American Society of Brewing Chemists, St. Paul, MN, U.S.A, 2009, http://dx.doi.org/10.1094/ ASBCMOA-Hops-16, Approved 1999, rev. [7] A.J.P. Hofte, R.A.M. Van der Hoeven, S.Y. Fung, R. Verpoorte, U.R. Tjaden, J. Van der Greef, Characterization of hop acids by liquid chromatography with negative electrospray ionization mass spectrometry, J. Am. Soc. Brew. Chem. 56 (1998) 118–122. [8] B. Jaskula, K. Goiris, G. Rouck, G. Aerts, L. Cooman, Enhanced quantitative extraction and HPLC determination of hop and beer bitter acids, J. Inst. Brew. 113 (2007) 381–390. [9] M.G. Malowicki, T.H. Shellhammer, Isomerization and degradation kinetics of hop (Humulus lupulus) acids in a model wort-boiling system, J. Agric. Food Chem. 53 (2005) 4434–4439. [10] R.A. Aitken, A. Bruce, J.O. Harris, J.C. Seaton, Quantitative analysis of beer bittering substances by thin-layer chromatography, J. Inst. Brew. 73 (1967) 528–534. [11] R.A. Aitken, A. Bruce, J.O. Harris, J.C. Seaton, Quantitative analyses of beer bittering substances and hop resins by thin-layer chromatography, J. Inst. Brew. 74 (1968) 436–440. [12] R. Franiau, R. Mussche, Quantitative determination of hop bitter substances and their derivatives in hop extracts by thin layer chromatography, J. Inst. Brew. 80 (1974) 59–67. [13] G.T. Winter, J.A. Wilhide, W.R. LaCourse, Molecular ionization-desorption analysis source (MIDAS) for mass spectrometry: thin-layer chromatography, J. Am. Soc. Mass Spectrom. 27 (2016) 352–358. [14] L. Ceslova, M. Holcapek, M. Fidler, J. Drstickova, M. Lisa, Characterization of prenylflavonoids and hop bitter acids in various classes of Czech beers and hop extracts using high-performance liquid chromatography–mass spectrometry, J. Chromatogr. A 1216 (2009) 7249–7257. [15] M. Zachariasova, T. Cajka, M. Godula, A. Malachova, Z. Veprikova, J. Hajslova, Analysis of multiple mycotoxins in beer employing (ultra)-high-resolution mass spectrometry, Rapid Commun. Mass Spectrom. 24 (2010) 3357–3367. [16] T. Cajka, K. Riddellova, M. Tomaniova, J. Hajslova, Ambient mass spectrometry employing a DART ion source for metabolomic fingerprinting/profiling: a powerful tool for beer origin recognition, Metabolomics 7 (2011) 500–508. [17] ASBC Methods of Analysis, Online. Hops Method 6. ␣- and - Acids in Hops and Hop Pellets by Spectrophotometry and by Conductometric Titration, American Society of Brewing Chemists, St. Paul, MN, U.S.A, 2008, http://dx. doi.org/10.1094/ASBCMOA-Hops-6, Approved 1959, Rev. [18] H.O. Askew, Separation of humulones and isohumulones in aqueous buffers, J. Inst. Brew. 71 (1965) 421–431.
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Full Length Article
Analysis of hop acids by thin-layer chromatography and the Molecular Ionization Desorption Analysis Source (MIDAS) for mass spectrometry Gregory T. Winter, Joshua A. Wilhide, William R. LaCourse ∗ University of Maryland, Baltimore County, 1000 Hilltop Circle, Baltimore, MD, 21250, USA
a r t i c l e
i n f o
Article history: Received 14 July 2017 Received in revised form 21 August 2017 Accepted 22 August 2017 Available online xxx Keywords: Ambient ionization Hop acids MIDAS Thin-layer chromatography
a b s t r a c t A thin-layer chromatography ambient mass spectrometry method was developed for the quantitative analysis of hop acids. Hop acids were extracted from three varieties of hop pellets, Apollo, Cluster and Czech Saaz. Reversed-phase thin-layer chromatography provided the optimum stationary phase for separation and ionization. Using standards, a method was developed to separate ␣-, iso-␣- and -acids by thin-layer chromatography. Each thin-layer chromatography plate was analyzed directly by the Molecular Ionization Desorption Analysis Source (MIDAS). MIDAS is an atmospheric pressure ambient ionization source for mass spectrometry. Quantitative analysis of the hop extracts was made by successive analysis of known amounts of standards and the determination of a response factor. The expected trend of total ␣-acid content with Apollo > Cluster > Czech Saaz was observed. Variation in the results was attributed to manual TLC plate spotting and not to the ionization source. © 2017 Elsevier B.V. All rights reserved.
1. Introduction Hops are the flowers of the female variety of the plant Humulus lupulus [1]. Hops are used in the beer brewing process to add bitterness and aroma to the final product. While there are a variety of compounds that contribute to the aromatic flavors in beer, there are a few primary compounds that contribute bitterness. These compounds, known as alpha acids (␣-acids), are humulone, adhumulone and cohumulone. In their native form, however, the ␣-acid compounds are not readily soluble in water and do not contribute any bitter flavor [2]. When hops are added during the boiling process used in making beer, the ␣-acids undergo thermal isomerization, via an acyloin-type ring contraction, to the more water soluble, bitter tasting, iso-␣-acids [2]. Also found in hops are three primary beta acids (-acids) including lupulone, adlupulone and colupulone. The oxidation products of these acids, such as hulupinic acid, can contribute additional bitterness to the final product [1]. Determining the content of ␣- and -acids in a sample of hops, whether they be whole leaf, pellet or extract is of primary importance in the brewing industry. Many validated methods exist for this purpose, primarily using High Performance Liquid Chromatography (HPLC) with UV–vis detection [3–6]. In addition to these validated methods, some researchers have developed their own [7–9].
In this paper, thin-layer chromatography (TLC) is used to separate ␣- and -acids extracted from hop pellets. TLC offers the advantage of being simple and cost effective, using very little solvent. TLC has been used for the separation of ␣- and -acids previously [10–12]. Typically, in order to identify a spot on a TLC plate, the plate either needs to be stained with a particular reagent or the spot removed and analyzed in a separate experiment. Presented here is the direct analysis of ␣- and -acids extracted from hop pellets from the surface of a TLC plate using the Molecular Ionization Desorption Analysis Source (MIDAS). MIDAS is a recently introduced ambient ionization and sampling platform for mass spectrometry [13]. Previous characterization of beer and hops has employed ambient ionization, including atmospheric pressure chemical ionization (APCI) [14] and direct analysis in real time (DART) [15,16]. For comparison and to establish a reference value for the acid content in each hop sample, ASBC method HOP-6A was used [17]. This method is a UV–vis spectrophotometric method for determining total ␣- and -acid content in hop samples. The goal of these experiments is to assess the potential for using TLC and MIDAS for quantitative determination of ␣- and -acids from hop pellets.
2. Materials and methods 2.1. Standards and materials
∗ Corresponding author. E-mail address: [email protected] (W.R. LaCourse).
Methanol, toluene, sodium hydroxide, formic acid and phosphoric acid (85%) were purchased from Fisher Scientific (Pittsburgh,
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Fig. 1. Structures of ␣, iso-␣, and  acids.
PA). C18 modified silica gel 60 plates, 5.0 × 10.0 cm, 250 m thickness, (EMD Millipore, Billerica, MA) were used for ␣-, iso-␣- and -acid separation. Additional testing was done on normal-phase silica gel 60 plates, 2.5 × 7.5 cm, 250 m thickness (EMD Millipore, Billerica, MA). Certified standards, International Calibration Standard I3 (ICSI3) and International Calibration Extract 3 (ICE-3) were purchased from the American Society of Brewing Chemists (St. Paul, MN). ICS-I3 contains the trans form of the dicyclohexylamine complex (DCHA)-iso-␣-acid of humulone, adhumulone and cohumulone (62.3% w/w). ICE-3 contains the un-isomerized forms of humulone, adhumulone and cohumulone (44.64% w/w), as well as the -acids lupulone, adlupulone, and colupulone (24.28% w/w). Structures of these compounds can be found in Fig. 1. Pelletized hops of the Apollo, Cluster, and Czech Saaz variety were purchased from a local homebrew supply store. These varieties were chosen as they span the low, medium and high ␣-acid content range, respectively. As stated on the packaging, the Apollo, Cluster and Czech Saaz varieties contained 17%, 8.1% and 2.6% ␣-acid with -acid content of 6.7%, 5.2% and 4.7%, respectively. 2.2. Standards preparation To prepare the ICE-3 standard, 550.8 mg was placed in 20.00 mL of methanol and sonicated until dissolved. This solution was then diluted to a final volume of 100.00 mL with methanol. Based on the certified concentration, this dilution results in 0.597 mg mL−1 of lupulone and adlupulone, 0.740 mg mL−1 of colupulone, 1.694 mg mL−1 humulone and adhumulone and 0.765 mg mL−1 of cohumulone. ICS-I3 was dissolved to a concentration of 1.300 mg mL−1 in methanol. 2.3. Hop extract preparation Hop extracts prepared using this method were used for both the UV and TLC experiments. Hop pellets were ground until the entire sample could be passed through a number 20 stainless steel sizing screen. 50.00 mL of toluene was then added to 2.5000 (±0.0015) g of ground hop pellets in an Erlenmeyer flask. The flask was cov-
ered in foil to protect against light and shaken for 30 min. After shaking, the sample was centrifuged at 1800 rpm for five minutes. The supernatant was then collected for analysis. All samples were stored at −20 ◦ C under a nitrogen atmosphere until analysis. These compounds are known to be light, heat and oxygen sensitive and care should be taken to minimize their exposure to the atmosphere during preparation and storage. 2.4. UV method To prepare the blank, 5.00 mL of toluene was added to a 100.00 mL volumetric flask and brought to volume with methanol, creating dilution A. To prepare dilution B, 3.00 mL of dilution A was added to a 50.00 mL volumetric flask and brought to volume with alkaline methanol. To prepare alkaline methanol, 0.20 mL of 6.0 N sodium hydroxide was added to 100.00 mL of methanol. Each hop extract sample was prepared in the same manner as the blank, with the initial 5.00 mL being the hop extract. After preparation, samples were run immediately on a JASCO V560 spectrophotometer (Easton, MD). The instrument was run in scanning mode from 270 to 450 nm. Each sample was analyzed five times with fresh sample placed in the cuvette for each analysis. 2.5. Thin-layer chromatography-MIDAS method For method development, four L of a 1:1 mixture of ICS-I3 and ICE-3 was spotted onto the TLC plates. Plates were developed in either 100% methanol; 1% phosphoric acid in methanol (v/v); or 1% formic acid in methanol (v/v). For quantitative analysis of both the standards and sample, 1.0 L of each was manually spotted onto a TLC plate using a glass microsyringe. The plate was then developed in a 1% solution of formic acid in methanol (v/v). Due to the high concentration of ␣-acids in the Apollo and Cluster samples, these solutions were further diluted 1:1 (v/v) in methanol prior to analysis. 2.6. Mass spectrometer Mass spectrometer tuning parameters were the same as those used previously [1]. Briefly, analyses were performed on
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Table 1 ␣ and -acid content by technique including original package values. Hop Variety
Technique
(w/w%) ␣-acid content (±95% CI)
(w/w%) -acid content (±95% CI)
Ratio Total ␣: acid content (%RSD)
Apollo
UV–vis TLC-MIDAS Package
18.17 (0.12) 17.7 (7.1) 17
4.85 (0.10) 6.2 (1.9) 6.7
3.75 (2.20) 2.66 (6.89)a 2.5
Cluster
UV–vis TLC-MIDAS Package
7.31 (0.05) 3.7 (0.9) 8.1
4.18 (0.04) 3.8 (0.7) 5.2
1.75 (1.39) 0.88 (20.10)a 1.6
Czech Saaz
UV–vis TLC-MIDAS Package
2.84 (0.03) 1.9 (0.5) 2.6
4.44 (0.03) 7.4 (1.3) 4.7
0.64 (1.46) 0.22 (2.65)a 0.6
a
Ratios were determined from an average of absolute peak areas, not from values determined by response factors.
(w/w%)˛-acidcontent = d × (−51.56A355 + 73.79A325 − 19.07A275 )
(2)
(w/w%)ˇ-acidcontent = d × (−55.57A355 + 47.59A325 + 5.10A275 )
(3)
Table 1 contains the average ␣- and -acid content calculated for each of the three hop varieties with the associated 95% confidence interval (CI). 3.2. Hop acid separation and MIDAS method development
Fig. 2. UV absorbance spectra for Apollo, Cluster and Czech Saaz hop varieties.
a PerkinElmer AxION Time-of-Flight mass spectrometer operated in negative ion mode. Negative mode parameters include capillary exit, skimmer, RF and offset voltages of −100 V, −20 V, −450 V and −10.3 V, respectively. Spectra were acquired at a rate of 2 spectra s−1 . 2.7. MIDAS parameters Nitrogen gas temperature was maintained at 180 ◦ C with a supply pressure of 40 psi and a purity of >99%. The corona discharge was operated with a negative polarity at −2.5 kV and 12 A. Atmospheric relative humidity ranged between 18 and 50%. No additional reagents or solvents were added to the system. For all analyses, the TLC plate was moved through the sampling region at a rate of 0.3 mm s−1 . 3. Results and discussion 3.1. UV experiment Each hop sample produced a similar UV spectrum with the absorbance intensity increasing with greater ␣- and -acid content. Fig. 2 contains an example UV absorbance spectrum for each of the three hop varieties tested. The absorbance maxima at the wavelengths of 355, 325 and 275 nm are used to calculate the ␣ and  acid content in each sample. There are three equations contained within the method. Eq. (1) accounts for the dilution of the sample, Eq. (2) calculates the ␣-acid content, and Eq. (3) calculates the -acid content. d=
vol.dil.A (mL) × voldil.B (mL) 500 ∗ aliq.extract (mL) × aliq.dil.A (mL)
(1)
To ensure that, if present, any iso-␣-acids would be detected, it was important that the TLC method successfully separate the iso-␣acids from the ␣-acids, as the acid isomers have the same molecular weight and cannot be distinguished by mass spectrometry alone. Separating the iso-␣-acids from the ␣-acids may also allow this method to be used for finished beer. Historically, TLC separations involving ␣- and -acids used normal-phase plates [10–12]. For this application, however, no ionization was achieved when testing these compounds on normal-phase plates. The lack of ionization is likely due to significant non-covalent bonding of the analyte molecules to the stationary phase of the TLC plate. Therefore, reversed-phase TLC plates were used. These plates also have the advantage of employing solvent conditions similar to those used in HPLC experiments, making method development more straightforward. Initially, a 1% solution of phosphoric acid in methanol (v/v) was used as the mobile phase. Phosphoric acid was chosen as it is a common modifier used in HPLC separations of these compounds [8]. It was not expected that a modifier would adversely impact ionization or detection because there was no direct introduction of the mobile phase to the instrument, unlike in traditional HPLC techniques. Under these solvent conditions, three distinct groups of compounds corresponding to the ␣-, iso-␣- and -acids were found when observing the TLC plate under a UV lamp. Complete separation of each subset of compounds is not required as each mass can be extracted individually from the total ions observed, providing sufficient information to determine the total amount of each type of compound present. While some ionization of each acid was observed, signal intensity was inconsistent and not reproducible. The likely cause of the inconsistent ionization was the phosphoric acid used in the mobile phase. Phosphoric acid remained on the TLC plate after the plate had dried. The result of the remaining phosphoric acid was the generation of phosphoric acid clusters when the plate was scanned using the MIDAS. With successive scans of the TLC plates, the intensity of the phosphoric acid clusters continued to increase, indicating that carryover is a problem when using this method. The increase in ion background resulted in suppression of the analyte signal. Therefore, phosphoric acid was removed and the plate developed using 100% methanol as the mobile phase. With this solvent, however, the ␣and iso-␣-acids failed to separate. An extracted ion chromatogram (EIC) of this experiment can be found in Fig. 3A.
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Fig. 3. (A) Extracted ion chromatogram of a mixture of ICE-3 and ICS-I3 developed using 100% methanol (B) extracted ion chromtatogram of a mixture of ICE-3 and ICS-I3 developed in a 1% solution of formic acid in methanol (v/v).
Fig. 4. Example mass spectra for: (A) humulone, adhumulone, isohumulone and isoadhumulone, (B) cohumulone and isocohumulone, (C) lupulone and adlupulone, (D) colupulone.
The failure to adequately separate the compounds results from the lack of an acidic mobile phase. There is not enough difference in polarity between the ␣- and iso-␣-acids in 100% methanol to separate effectively on a 10 cm TLC plate. A 1% solution (v/v) of phosphoric acid in methanol has a pH of approximately 2 at room temperature. The pKa’s of the ␣-acids are in the range of 4.0–5.5, while the iso-␣-acids are in the range of 5.5–7.8 [18,7]. Thus, at the pH of the phosphoric acid mobile phase, the ␣-acids will be nearly
fully protonated, and therefore more non-polar, while the iso-␣acids will have a mixture of protonated and ionized species. This difference in protonation allows for the effective separation of the two groups on the TLC plate. In order to maintain a similar pH while minimizing interferences, formic acid was chosen to replace the phosphoric acid. A 1% solution (v/v) of formic acid in methanol results in a pH of 2.2 at room temperature. Formic acid is a volatile acid that is used as a
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modifier in HPLC–MS experiments. Due to its volatility, any formic acid remaining on the plate after it had dried was minimal, resulting in a cleaner background with no clusters observed. A mixture of ICE-3 and ICS-I3 was developed on a plate using a 1% solution (v/v) of formic acid in methanol. An EIC of this separation can be found in Fig. 3B. Using formic acid, the three groups of compounds were successfully separated. Example mass spectra obtained for each acid, observed as the deprotonated species [M−H]− , can be found in Fig. 4. As many of these compounds are isomeric, they are observed at the same mass to charge (m/z) ratio with the only distinguishing factor being the different retention times determined from the TLC separation.
3.3. Hop sample analysis In order to quantify the amount of each acid contained in each hop sample a response factor based on the ICE-3 standard was first determined. The response factor was determined by analyzing a known quantity of standard that had been spotted and developed on the plate. The response factor was calculated as the peak area divided by the amount of standard on the plate (in mg). The concentration in each sample was then calculated by multiplying the peak area obtained from the unknown by the response factor and any appropriate dilution factors. In an attempt to compensate for any variability between TLC plates, each plate was cut in half. Three replicates of standards were run on one half while three replicates of samples were run on the other. Each sample therefore was analyzed six times, while a total of 18 standard replicates were analyzed. The total ␣- and -acid content in each sample was calculated based on the average response factor determined from the corresponding standard run. Example EIC’s obtained for the analysis of Apollo, Cluster and Czech Saaz hop varieties are contained in Fig. 5. The calculated ␣and -acid contents with corresponding 95% confidence interval (CI) are contained in Table 1. It is worth noting that it took approximately 20–25 min to develop each plate and only four minutes to analyze each spot using MIDAS. Therefore, a single plate containing three replicates can be developed and analyzed in the time it takes to obtain a single chromatogram by conventional HPLC methods, offering savings in terms of time and solvent cost. Variation was observed in the response factors determined for the standards. When peak areas for each of the 18 acid standard trials were compared, relative standard deviation (RSD) values ranged from 38 to 51%. RSDs determined for the individual samples were also high, ranging from 16 to 35%. The tabulated results show that semi-quantitative data is being produced using this method, as the results follow the expected trend of ␣-acid content. There is, however, too much variation for the data to be considered reliable for full quantitation. Encouragingly, the variation can be attributed to the manual TLC plate spotting technique. Evidence of this was found when comparing the ratios of peak areas for total ␣: acid content. While the RSDs for total ␣- and - acid peak areas determined from the 18 replicates of the standard ICE-3 were 50.17% and 40.39% respectively; the ratio of total ␣: was found to be 1.67 with an RSD of 10.45%. The calculated ratio based on the certified values in the ICE-3 standard is 1.84. This result indicates that despite the variation in absolute peak area, the ratio remains consistent, pointing to inconsistencies in plate spotting. For the three hop samples, the ratios of total ␣: acid, as determined by total peak area (not response factor) for Apollo, Cluster and Czech Saaz are included in Table 1. Also included in Table 1 are the original package values for reference. Comparison with the package values is difficult as no certificate of analysis is available and it is unknown how the package values were obtained.
Fig. 5. EICs obtained for extracts of (A) Apollo (B) Cluster and (C) Czech Saaz hop varieties.
Deviation from the values obtained using the UV–vis method could result from bias within the method due to interfering species present in the whole hop extract. Additionally, the TLC-MIDAS still may suffer from incomplete or inconsistent ionization, but it is difficult to tell from these results given the variation in plate spotting.
4. Conclusions While this current experiment is focused on extracts from hop pellets containing ␣- and -acids, this method has been shown to successfully separate the iso-␣-acids from the un-isomerized
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forms. Therefore, this method could be extended to the analysis of isomerized hop extracts and potentially to finished beer. The goal of these experiments was to determine the feasibility of using TLC and MIDAS analysis to quantify the ␣- and -acid content in hop pellets. The accepted UV method was run in order to obtain values for the actual ␣- and -acid content in each sample, ensuring that a known value was used for comparison with the mass spectrometry results and not an assumed value from packaging, as product degradation is possible depending on storage time and conditions. The MIDAS-TLC method shows semi-quantitative results, with the determined ␣-acid content for each hop variety following the expected trend of Apollo > Cluster > Czech Saaz. While there was wide variation in the raw data, the lower RSDs found within the ratio of total ␣: content in the standards as well as the samples indicates that with further development of the TLC spotting method, fully quantitative results and an effective comparison between methods should be possible. Using a suitable internal standard may eliminate problems relating to manual plate spotting. Alternatively, the use of an automatic TLC spotter could be explored. Funding This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors. Acknowledgements The authors would like to acknowledge Ian Shaffer of the Molecular Characterization and Analysis Complex at UMBC for technical support and Maggie LaCourse for editing and review. References [1] S. Hieronymus, For the Love of Hops, vol. 178, Brewers Publications, Boulder, Colorado, 2012, pp. 19. [2] B. Jaskula-Goiris, G. Aerts, L. De Cooman, Hop ␣-acids isomerisation and utilisation: an experimental review, Cerevisia 35 (2010) 57–70. [3] ASBC Methods of Analysis, Online. Hops Method 9. ␣- and - iso-␣-Acids by High-Performance Liquid Chromatography, American Society of Brewing Chemists, St. Paul, MN, U.S.A, 2008, http://dx.doi.org/10.1094/ASBCMOAHops-9, Approved 1987, rev.
[4] ASBC Methods of Analysis, Online. Hops Method 14. ␣-Acids and - Acids in Hops and Hop Extracts by HPLC, American Society of Brewing Chemists, St. Paul, MN, U.S.A, 2008, http://dx.doi.org/10.1094/ASBCMOA-Hops-14, Approved 1990, rev. [5] ASBC Methods of Analysis, Online. Hops Method 15. Iso-␣-Acids in Isomerized Hop Pellets by HPLC, American Society of Brewing Chemists, St. Paul, MN, U.S.A, 2008, http://dx.doi.org/10.1094/ASBCMOA-Hops-15, Approved 1993, rev. [6] ASBC Methods of Analysis, Online. Hops Method 16. Iso- ␣ –, ␣-, and - Acids in Hop Extracts and Isomerized Hop Extracts by HPLC, American Society of Brewing Chemists, St. Paul, MN, U.S.A, 2009, http://dx.doi.org/10.1094/ ASBCMOA-Hops-16, Approved 1999, rev. [7] A.J.P. Hofte, R.A.M. Van der Hoeven, S.Y. Fung, R. Verpoorte, U.R. Tjaden, J. Van der Greef, Characterization of hop acids by liquid chromatography with negative electrospray ionization mass spectrometry, J. Am. Soc. Brew. Chem. 56 (1998) 118–122. [8] B. Jaskula, K. Goiris, G. Rouck, G. Aerts, L. Cooman, Enhanced quantitative extraction and HPLC determination of hop and beer bitter acids, J. Inst. Brew. 113 (2007) 381–390. [9] M.G. Malowicki, T.H. Shellhammer, Isomerization and degradation kinetics of hop (Humulus lupulus) acids in a model wort-boiling system, J. Agric. Food Chem. 53 (2005) 4434–4439. [10] R.A. Aitken, A. Bruce, J.O. Harris, J.C. Seaton, Quantitative analysis of beer bittering substances by thin-layer chromatography, J. Inst. Brew. 73 (1967) 528–534. [11] R.A. Aitken, A. Bruce, J.O. Harris, J.C. Seaton, Quantitative analyses of beer bittering substances and hop resins by thin-layer chromatography, J. Inst. Brew. 74 (1968) 436–440. [12] R. Franiau, R. Mussche, Quantitative determination of hop bitter substances and their derivatives in hop extracts by thin layer chromatography, J. Inst. Brew. 80 (1974) 59–67. [13] G.T. Winter, J.A. Wilhide, W.R. LaCourse, Molecular ionization-desorption analysis source (MIDAS) for mass spectrometry: thin-layer chromatography, J. Am. Soc. Mass Spectrom. 27 (2016) 352–358. [14] L. Ceslova, M. Holcapek, M. Fidler, J. Drstickova, M. Lisa, Characterization of prenylflavonoids and hop bitter acids in various classes of Czech beers and hop extracts using high-performance liquid chromatography–mass spectrometry, J. Chromatogr. A 1216 (2009) 7249–7257. [15] M. Zachariasova, T. Cajka, M. Godula, A. Malachova, Z. Veprikova, J. Hajslova, Analysis of multiple mycotoxins in beer employing (ultra)-high-resolution mass spectrometry, Rapid Commun. Mass Spectrom. 24 (2010) 3357–3367. [16] T. Cajka, K. Riddellova, M. Tomaniova, J. Hajslova, Ambient mass spectrometry employing a DART ion source for metabolomic fingerprinting/profiling: a powerful tool for beer origin recognition, Metabolomics 7 (2011) 500–508. [17] ASBC Methods of Analysis, Online. Hops Method 6. ␣- and - Acids in Hops and Hop Pellets by Spectrophotometry and by Conductometric Titration, American Society of Brewing Chemists, St. Paul, MN, U.S.A, 2008, http://dx. doi.org/10.1094/ASBCMOA-Hops-6, Approved 1959, Rev. [18] H.O. Askew, Separation of humulones and isohumulones in aqueous buffers, J. Inst. Brew. 71 (1965) 421–431.
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