Unified Flavor Quantitation: Toward High-Throughput Analysis of Key Food Odorants and Tastants by Means of Ultra-High-Performance Liquid Chromatography Tandem Mass Spectrometry

Article abstract of DOI:10.1021/acs.jafc.9b03466

Because foods are perceived through combined inputs from taste and odor, which are determined by the concentration of the individual odor and taste molecules, the unified high-throughput quantitation of volatile odorants and non-volatile tastants with the

Full text of DOI:10.1021/acs.jafc.9b03466

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Chemistry and Biology of Aroma and Taste
Unified Flavor Quantitation: Towards High-Throughput Analysis
of Key Food Odorants and Tastants by Means of UHPLC-MS/MS
Christoph Hofstetter, Andreas Dunkel, and Thomas Hofmann
J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.9b03466 • Publication Date (Web): 09 Jul 2019
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Journal of Agricultural and Food Chemistry
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Unified Flavor Quantitation: Towards High-Throughput
Analysis of Key Food Odorants and Tastants by Means of
UHPLC-MS/MS
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†
#‡
† #‡
Christoph Konrad Hofstetter , Andreas Dunkel and Thomas Hofmann
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†Chair of Food Chemistry and Molecular and Sensory Science, Technical University
of Munich, Lise-Meitner-Str. 34, D-85354 Freising, Germany,
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#Leibniz-Institute for Food Systems Biology at the Technical University of Munich,
Lise-Meitner-Str. 34, D-85354 Freising, Germany, and
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‡Bavarian Center for Biomolecular Mass Spectrometry, Technical University of
Munich, Gregor-Mendel-Straße 4, D-85354 Freising, Germany.
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*
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To whom correspondence should be addressed
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PHONE
+49-8161/71-2902
FAX
+49-8161/71-2949
E-MAIL
thomas.hofmann@tum.de
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Abstract
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As foods are perceived through combined inputs from taste and odor, which are
determined by the concentration of the individual odor and taste molecules, the unified
high-throughput quantitation of volatile odorants and non-volatile tastants with the very
same instrumental set-up has been a long-standing, but yet unmet dream. The
research presented here for the first time demonstrates, after only minimal sample
work-up, the highly accurate, rapid, and sensitive unified quantitation of odorants and
tastants of key flavour molecules in apple juice on a single UHPLC-MS/MS platform
over a large dynamic range of up to 6 orders of magnitude. While flavor active
aldehydes, ketones and organic acids were analyzed after derivatization with 3-
nitrophenylhydrazine, taste-active polyphenols and odor-active esters were directly
analyzed by means of UHPLC-MS/MS with and without target analyte enrichment
through stir-bar sorptive extraction. This “unified flavor quantitation” approach holds
promise to accelerate the transition of today’s labor and time consuming, low-
throughput analysis of odorants and tastants into a new era of high-performance
quantitation of key flavor molecules.
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Key words: taste, aroma, odorants, apple juice, LC-MS, unified flavor quantitation
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Introduction
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The health and environmental challenges arising from modern lifestyles call for
avantgardistic scientific approaches helping to better understanding consumers’
decisions on food choice and targeting food engineering to deliver healthy nutrition
without compromising aroma and taste and authentic eating experiences with positive
impact on the environment and food safety. To overcome flavor defects in food
products, induced by reducing levels of highly palatable ingredients (salt, sugar, fat
etc.), by adding health-promoting phytometabolites, or by using alternative raw
materials (plant-based proteins, algae-based lipids etc.), respectively, the consumer
acceptance of such products depends on new solutions and technologies capable of
fine-tuning flavor deviations for the delivery of truly authentic flavor signatures. To
achieve this, a thorough understanding of how our chemical senses olfaction and taste
deconvolute our foods’ puzzling world of odorants and tastants is of prime importance.
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In contradiction to traditional views, application of the principle of bioresponse-
guided identification to the discovery of volatile odorants and non-volatile tastants by
means of aroma extract dilution analysis1,2 and taste dilution analysis3,4 has
demonstrated the sheer unlimited variations in food flavors to be created by a
“combinatorial chemosensory code” of a limited number of key molecules. Food odor
recognition has been shown to consult a large repertoire of ~380 odorant receptors to
sense a surprisingly small center group of 3 - 40 key food odorants per food item out
of a total of ~230 key food odorants among the 10.000 food-born volatiles identified.5
Complementing the constructive sense of olfaction, a small number of only ~30 taste
receptors is designed to sense chemically diverse non-volatiles, among which 15 - 40
per food item are “analytically”, that means without any further combinatorial
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processing, translated into the perception of the five basic taste modalities bitter,
sweet, sour, salty, and umami.6
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Most impressively, minimal chemosensory recombinants of 3 - 40 key odorants
and 15 - 40 key tastants have been demonstrated to be truly necessary and sufficient
for constructing the authentic percept of a specific food’s flavor in our brain such as,
e.g. black tea,7,8 prawns,9,10 and red wine . Therefore, the key mechanism by which
the brain encodes perceptual representations of behaviorally relevant food items is
considered through the synthesis of combinatorial chemosensory inputs into a unique
perceptual experience (“flavor object”), rather than through individual molecules.12–14
Within the recent decades, most key odorants and basic taste compounds have been
already successfully identified and, within a food category, small quantitative rather
than chemical structure variations in odorant/tastant codes were concluded to trigger
perceived differences in sensory phenotypes of a food category.5,15 Therefore,
changes in odor and taste profiles from the raw materials through the various
manufacturing process intermediates all the way up-stream to the consumer’s plate
may be monitored by high-precision and high-throughput mass spectrometric profiling
of the entire population of a given food’s key flavor compounds, coined
“sensometabolome”.16,17
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In order to counteract the analytical challenges in accurate quantitation of
odorants and tastants, which are largely differing in concentration, volatility, and
chemical stability, the so-called stable isotope dilution analysis (SIDA) has been
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successfully introduced in flavor research, using stable isotope ( C, H)-labeled
analogue molecules of the odorants and tastants as most suitable internal standards
for GC-MS and LC-MS analysis.11,18–21
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Although the analysis of odor-active volatiles by means of high-resolution GC-
MS, GC/GC-MS, and GC˟GC-MS, respectively, is highly accurate and sensitive, gas
chromatographic analysis is limited to volatile analysis and, therefore, usually requires
time-consuming separation of the volatile fraction from non-volatiles prior to analysis,
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e.g. by solvent extraction and SAFE distillation. Another challenge in odorant analysis
is the large concentration range of odorants in food that need to be captured
analytically. Using the earlier reported meta-analytical data on 230 key food odorants,
which by definition exceed their threshold concentration in 227 food samples
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analyzed, the concentrations of these key food odorants, grouped in different chemical
classes, are plotted against their human threshold concentration in Figure 1. This plot
indicates that the key food odorants, depending on their chemical class and individual
threshold concentration, are present in a sensorially relevant concentration range of
~1 pmol/kg as found for thiols and sulphides, respectively, all the way up to ~10-100
mmol/kg as typical for alcohols. In consequence, odorant quantitation by means of GC-
MS requires that different sample volumes are worked up or that the aroma extract
prepared is injected several times at different dilutions in order to account for the
narrow linear range of four orders of magnitude for standard GC-MS systems.
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In comparison, analysis of non-volatile tastants is typically performed by liquid
chromatography hyphenated to a mass spectrometer. Sensorially relevant levels of
taste active compounds, that means by definition concentrations in foods exceeding
their detection threshold for primary taste activity or taste enhancement, are in the
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µmol/kg range, as observed for astringent/bitter polyphenols and terpenoids as well
as taste enhancers,24,16,10,25 and in the mmol/kg range as typically found for basic taste
compounds such as, e.g. amino acids, organic acids, and carbohydrates,
respectively.24,16,10,25
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As foods are perceived through combined inputs from taste and odor, which are
determined by the concentration of the individual odor and taste molecules, the unified
high-throughput quantitation of volatile odorants and non-volatile tastants with the very
same instrumental set-up would be highly desirable. New developments in ultra-high
performance liquid chromatography (UHPLC) and fast-scanning mass spectrometers
now hold promise to enable, after minimal sample work-up, the highly accurate and
sensitive unified quantitation of odorants and tastants on a single instrument platform
over a large dynamic range of up to 6 orders of magnitude.
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The objective of the present study was, therefore, to investigate the unified
UHPLC-MS/MS quantitation of key odorants and tastants in foods using apple juice as
an example. Based on previous research, E-2-hexenal (1), hexanal (2), β-
damascenone (3), acetaldehyde (4), ethyl butanoate (5), butyl acetate (6), ethyl 2
methylbutanoate (7), ethyl 2 methylpropanoate (8), ethyl propanoate (9), hexyl acetate
(10), pentyl acetate (11), methyl 2 methylbutanoate (12), propyl 2-methylbutanaote
(13), and ethyl hexanoate (14) are considered key odorants in apple juice (Figure 3),26–
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and, therefore, should be analyzed in the following study. In addition, the unified
quantitation method should comprise the sweet carbohydrates glucose (15) and
fructose (16), the sour tasting organic acids malic acid (17) and citric acid (18), the
sweet tasting polyphenol phlorizin (19), as well as the key astringent polyphenols
epicatechin (20) and chlorogenic acid (21).30–33
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Chemicals. The following compounds were obtained commercially: 3-
nitrophenylhydrazine hydrochloride, N-(3-dimethylaminopropyl)-N’-ethylcarbodiimide
hydrochloride, acetic acid, malic acid, citric acid, glucose, phlorizine, chlorogenic acid,
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(-)-epicatechine, glucose- C , malic acid-d , citric acid-1,5- C , chlorogenic acid ethyl
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ester-d (Sigma-Aldrich, Steinheim, Germany), pyridine (Merck, Darmstadt, Germany),
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E-2-hexenal, hexanal, β-damascenone, acetaldehyde, ethyl hexanoate, butyl acetate,
methyl 2-methylbutanoate, ethyl 2-methylbutanoate, hexyl acetate, ethyl butanoate
(Givaudan, Vernier, Suisse), dihydrorobinetin (Extrasynthèse, Genay Cedex, France),
E-2-hexenal-d , β-damascenone-d , hexanal-d , ethyl hexanoate-d , butyl-d acetate,
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methyl 2-methyl-d butyrate, ethyl 2-methyl-d -butyrate, hexyl-d acetate, ethyl
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butyrate-d used as internal standards were purchased from aromaLAB AG (Planegg,
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Germany). Solvents used for HPLC-MS/MS analysis were of LC-MS grade (Honeywell,
Seelze, Germany). Acetonitrile-d used for qNMR was purchased from (Sigma-Aldrich,
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Steinheim, Germany). Water for chromatography was purified by the use of a Milli-Q
water advantage A 10 water system (Millipore, Molsheim, France). Triplicates from four
clear apple juice samples A, C, D and E, and two cloudy apple juice samples B and F
were purchased from a local retailor (Freising, Germany).
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Sample Preparation. Apple juice samples were membrane-filtered (Minisart
RC 15, 0.45 μm; Sartorius AG, Göttingen, Germany) and the following internal
standard mixture in acetonitrile/water (20/80, v/v) was added before further sample
work-up: E-2-hexenal-d (1-d , 1200 µg/kg), hexanal-d (2-d , 530 µg/kg), (2E)-β-
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damascenone-d (3-d , 7.4 µg/kg), acetaldehyde-d (4-d , 8000 µg/kg), ethyl butyrate-
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d (5-d , 44 µg/kg), butyl-d acetate (6-d , 840 µg/kg), ethyl 2-methyl-d butyrate (7-d ,
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24 µg/kg), ethyl propanoate-d (9-d , 35 µg/kg), hexyl-d acetate (10-d , 175 µg/kg),
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methyl 2-methyl-d butyrate (12-d , 7.2 µg/kg), ethyl hexanoate-d (14-d , 3.4 µg/kg),
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glucose- C (15- C , 15.5 g/kg), malic acid-d (17-d , 5.3 g/kg), citric acid- C (18-
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C , 63.1 mg/kg), dihydrorobinetin (22, 160.5 mg/kg), and caffeic acid ethyl ester-d5
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(23-d , 78.5 mg/kg), respectively.
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Quantitation of Flavor-Active Aldehydes, Ketones, and Organic Acids after
3-NPH Derivatization. Following a method reported for the analysis of short and
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branched fatty acids, aliquots (40 µL) of the filtered apple juice samples spiked with
the internal standards were mixed with a solution (20 µL) of 3-nitrophenyl hydrazine
(3-NPH; 200 mmol/L) in acetonitrile/water (50/50, v/v) and a solution (20 µL) of N-(3-
dimethylaminopropyl)-N’-ethylcarbodiimide (EDC, 120 mmol/L) in acetonitrile/water
(50/50, v/v) containing 6 % pyridine. After incubation for 30 min at 40 °C, the solutions
were made up with acetonitrile/water (50/50, v/v) to 1.0 mL and, then, an aliquot (1 µL)
was injected into the UHPLC-MS/MS system.
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Internal Standard Calibration Curve. A stock solution of E-2-hexenal (12.0
mg/L), hexanal (5.3 mg/L), acetaldehyde (80 mg/L), malic acid (52.9 mg/L), citric acid
(0.94 mg/L), and glucose (155.6 mg/L) was prepared in acetonitrile/water (50/50, v/v)
and the exact concentration of each reference compound verified by means of
quantitative NMR. This stock solution was diluted 1:2, 1:3, 1:5, 1:10, 1:50, 1:100,
1:1000 with acetonitrile/water (50/50, v/v). To each dilution the same amount of internal
Standard (IS) was added for an end concentration of 1200 µg/L E-2-hexenal, 530 µg/L
hexanal, 8 mg/L acetaldehyde, 5.3 g/L malic acid, 15.5 g/L glucose and 94 mg/L citric
acid. 40 µl of each standard solution were used for derivatization using the instructions
above. For recovery experiments, the analytes were spiked into an apple juice using
the concentration ranges of the internal standard calibration curve as triplicates. After
addition of internal standard solutions, the samples were prepared as detailed above.
For the determination of the limit of detection (LOD) and the limit of quantitation (LOQ)
the 1:1000 dilution was further diluted 1:10, 1:100 and 1:1000 and the signal to noise
ratio was measured using the MultiQuant software (Sciex, Darmstadt, Germany). The
LOD value was set to a signal to noise ratio of 3 and the LOQ to a signal to noise ratio
of 10, respectively.
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UHPLC-MS/MS Analysis of 3-NPH-Derivatives. The samples were separated
by means of a Nexera UHPLC (Shimadzu Europa GmbH, Duisburg, Germany)
consisting of two LC pump systems 30AD, a DGU-20A5 degasser, a SIL-30AC
autosampler, a CTO-30A column oven, and a CBM-20A controller, and equipped with
a 100 x 2.1 mm, 100 A, Kinetex 1.7 µm C18 column (Phenomenex, Aschaffenburg,
Germany). A gradient of 0.1 % formic acid in water (solvent A) and 0.1 % formic acid
in acetonitrile (solvent B) was used (0.4 mL/min): 0 min, 20 % B; 2 min 20 % B; 4 min,
100 % B; 6 min, 100 % B; 7 min, 20 % B; 10 min, 20 % B. Gradient SBSE: 0 min, 40
% B; 1.5 min, 40 % B; 4.5 min, 60 % B; 6 min, 100 % B; 7 min, 100 % B; 9 min, 40 %
B; 10 min, 40 % B. The UHPLC was connected to a QTRAP 6500 mass spectrometer
(Sciex, Darmstadt, Germany), controlled by the Analyst 1.6.2 software (Sciex), and
operated in the full-scan mode (ion spray voltage: -4500 V) with the following
instrument settings: curtain gas (35 V), temperature (450 °C), gas 1 (55 V), gas 2 (65
V), collision activated dissociation (-2 V), and entrance potential (- 10 V).
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Stir Bar Sorptive Extraction (SBSE) of Flavor Molecules. Prior to use,
PDMS-stir bars (10 mm in length, 0.5 mm film thickness; Gerstel GmbH & Co.KG,
Mühlheim, Germany) were thermally pre-treated for 1 h at 280 °C using the Tube
Conditioner TC 2 for (Gerstel GmbH & Co.KG). After cooling, the stir bars were placed
into a glass vial (20 mL), an aliquot (20 mL) of apple juice spiked with the internal
standard mixture was added and, then, the glass vials were put on a lab shaker (800
rpm) for 60 min at room temperature. Thereafter, the stir bars were washed with
deionized water (5 mL), placed into a glass vial (1.5 mL) containing acetonitrile (100
µL), followed by shaking (800 rpm) for 60 min. An aliquot (3 µL) of the organic solution
was then directly injected into the UHPLC-MS/MS system.
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Internal Standard Calibration Curve. A stock solution of ethyl hexanoate (34
µg/L), butyl acetate (8.4 mg/L), methyl 2-methylbutanoate (72 µg/L), ethyl 2-
methylbutanoate (0.24 mg/L), hexyl acetate (1.7 mg/L), ethyl butanoate (0.44 mg/L),
and β-damascenone (74 µg/L) was prepared in acetonitrile/water (50/50, v/v). This
stock solution was diluted 1:2, 1:3, 1:5, 1:10, 1:50, 1:100, 1:1000 with acetonitrile/water
(50/50, v/v). To each dilution the same amount of internal standard was added to reach
an end concentration of 3.4 µg/L for ethyl hexanoate, 840 µg/L for butyl acetate, 7.2
µg/L for methyl 2-methylbutanoate, 24 µg/L for ethyl 2-methylbutanoate, 175 µg/L for
hexyl acetate, 44 µg/L for ethyl butanoate, and 7.4 µg/L for β-damascenone,
respectively. Aliquots (20 mL) of each standard solution were used for SBSE as
detailed above. For recovery experiments, the analytes were spiked into an apple juice
using the concentration ranges of the internal standard calibration curve as triplicates.
After addition of the internal standards, the samples were prepared as reported above.
For the determination of the LOD and LOQ values the 1:1000 dilution was further
diluted 1:10, 1:100 and 1:1000 and the signal to noise ratio was measured using
MultiQuant software (Sciex, Darmstadt, Germany). The LOD value was set to a signal
to noise ratio of 3 and the LOQ to a signal to noise ratio of 10, respectively.
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UHPLC-MS/MS Analysis of Flavor Molecules after SBSE-Enrichment. The
samples obtained after SBSE treatment were separated by means of an ExionLC
(Sciex, Darmstadt, Germany), consisting of two LC pump systems ExionLC AD Pump,
an ExionLC degasser, an ExionLC AD autosampler, an ExionLC AC column oven, and
an ExionLC controller, and equipped with a 100 x 2.1 mm, 1.7 µm, 100 A Kinetex C18
column (Phenomenex, Aschaffenburg, Germany). A gradient of 0.1 % formic acid in
water (Solvent A) and 0.1 % formic acid in acetonitrile (solvent B) was used (0.4
mL/min): 0 min, 40 % B; 1.5 min, 40 % B; 4.5 min, 60 % B; 6 min, 100 % B; 7 min, 100
% B; 9 min, 40 % B; 10 min, 40 % B. The UHPLC was hyphenated with a QTRAP
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6500+ mass spectrometer (Sciex, Darmstadt, Germany), controlled by the Analyst
1.6.3 software (Sciex), and operated in the full-scan mode (ion spray voltage: 5500 V)
using the following instrument settings: curtain gas (35 V), temperature (450 °C), gas
1 (55 V), gas 2 (65 V), collision activated dissociation (2 V), and entrance potential (10
V). Data interpretation was performed by using MultiQuant software, Analyst 1.6.3
(Sciex, Darmstadt, Germany) and Microsoft Excel 2016.
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Quantitation of Polyphenols. Aliquots (1 µL) of the membrane-filtered apple
juice samples spiked with the added internal standards were directly injected into the
UHPLC-MS/MS system.
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Internal Standard Calibration Curve. A stock solution of phlorizin (175 mg/L),
chlorogenic acid (223 mg/L), and epicatechine (201 mg/L) was prepared in
acetonitrile/water (20/80, v/v). The exact concentration was verified by means of
qNMR. This stock solution was diluted 1:2, 1:5, 1:10, 1:20, 1:50, 1:100, 1:200, 1:500,
1:1000 with acetonitrile/water (20/80, v/v). Dihydrorobinetin (160 mg/L) was used as
the internal standard for the quantitation of epicatechine and phlorizin, caffeic acid ethyl
ester-d (171 mg/L; 22-d ) was used for quantitative analysis of chlorogenic acid. For
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recovery experiments, the analytes were spiked into an apple juice using the
concentration ranges of the internal standard calibration curve as triplicates. LC-
MS/MS analysis of polyphenols was performed using the parameters described above
for 3-NPH-derivatives. For the determination of LOD and LOQ values the 1:1000
dilution was further diluted 1:10, 1:100 and 1:1000 and the signal to noise ratio was
measured using MultiQuant software (Sciex, Darmstadt, Germany). The LOD value
was set to a signal to noise ratio of 3 and the LOQ to a signal to noise ratio of 10,
respectively.
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Quantitative H Nuclear Magnetic Resonance Spectroscopy (qNMR).
qNMR was recorded on a 400 MHz Avance III spectrometer (Bruker, Rheinstetten,
Germany) equipped with a Broadband Observe BBFO plus Probe (Bruker,
Rheinstetten, Germany). Acetonitrile-d3 (600 µL) was used as solvent and chemical
shifts are reported in parts per million relative to the acetonitrile-d solvent signal. Data
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processing was performed by using Topspin NMR software vers. 3.2 (Bruker,
Eheinstetten, Germany). Quantitative NMR spectroscopy (q-NMR) was performed as
reported earlier through calibration of the spectrometer by applying the ERETIC 2 tool
using the PULCON methodology.35
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In order to develop a new analytical approach for high-throughput and joint quantitation
of key odorants and tastants in apple juices, the odorants E-2-hexenal (1), hexanal (2),
β-damascenone (3), acetaldehyde (4), ethyl butanoate (5), butyl acetate (6), ethyl 2-
methylbutanoate (7), ethyl 2-methylpropanoate (8), ethyl propanoate (9), hexyl acetate
(10), pentyl acetate (11), methyl 2-methylbutanoate (12), propyl 2-methylbutanaote
(13), and ethyl hexanoate (14), as well as the taste-active compounds glucose (15),
fructose (16), malic acid (17), citric acid (18), phlorizin (19), epicatechine (20), and
chlorogenic acid (21) should be analyzed by means of a single UHPLC-MS/MS
plattform.
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Method Development and Validation Experiments. To enable a highly
sensitive MS-detection of flavor-active aldehydes and organic acids over a large
dynamic range and after only minimum sample work-up, samples of an apple juice was
derivatized upon incubation with 3-nitrophenyl hydrazine (3-NPH) for 15 to 120 min
and at temperatures between 20 and 60 °C in the presence of N-(3-
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dimethylaminopropyl)-N’-ethylcarbodiimide in acetonitrile/water and, then, directly
analyzed by means of UHPLC-MS/MS. The odorants E-2-hexenal (1), hexanal (2) and
acetaldehyde (4), as well as the taste compounds glucose (15), fructose (16), malic
acid (17), and citric acid (18) were detected and their identity confirmed by comparing
the retention times and MS spectra of the candidate analytes in the apple juice with
those of reference compounds, followed by co-chromatography. To optimize MS/MS
detection for high-sensitivity quantitation, standard solutions of these analytes were
derivatized, syringe-infused into the MS ion source, and software-assisted ramping of
ion source and ion path parameters in the negative electrospray ionization mode was
performed to maximize fragment ion abundance. Varying derivatization time (15 – 120
min) and temperature (20 – 60 °C) as well as sample volume (20 - 1000 µL) of apple
juice, followed by UHPLC-MS/MS analysis revealed that already the use of a low
sample volume of 40 µL and the gentle derivatization conditions (30 min, 40 °C) are
fully adequate for unequivocal detection and quantitation of E-2-hexenal (1), hexanal
(2), acetaldehyde (4), glucose (15), fructose (16), malic acid (17), and citric acid (18)
in apple juice. Quantitation of the analytes was performed in less than 10 min by means
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18- C as internal standards spiked to the apple juice samples prior to 3-NPH
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derivatization (Figure 4). Spiking apple juice samples with additional amounts of the
analytes, followed by derivatization and UHPLC-MS/MS analysis revealed good
recovery rates for the analytes ranging between 86 and 107 % (Table 1). The limit of
detection (LOD) and limit of quantitation (LOQ) were very low for the hydrophobic
odorants and ranged from 0.004 and 0.014 µmol/L for E-2-hexenal to 0.03 and 0.068
µmol/L for hexanal. In comparison, higher LOD and LOQ were found for the polar taste
compounds ranging from 0.05 and 0.15 µmol/L for citric acid to 23.7 and 83.3 µmol/L
for glucose (Table 1).
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Due to their low MS-sensitivity, a direct UHPLC-MS/MS analysis of the odor-
active esters were not possible without any enrichment step. Based on the suitability
of stir bar sorptive extraction (SBSE) for the enrichment of hydrophobic odorants prior
to HRGC-MS analysis,36 polydimethylsiloxane (PDMS) coated stir bars were added
into apple juice samples and, after incubation (15 – 120 min) at room temperature, the
stir bars were washed with deionized water and, then, the bound odorants extracted
with acetonitrile, followed by UHPLC-MS/MS analysis. The odor-active esters ethyl
butanoate (5), butyl acetate (6), ethyl 2-methylbutanoate (7), ethyl 2-methylpropanoate
(8), ethyl propanoate (9), hexyl acetate (10), pentyl acetate (11), methyl 2-
methylbutanoate (12), propyl 2-methylbutanaote (13), and ethyl hexanoate (14) as well
as β-damascenone (3) were detected and their identity confirmed by comparing the
retention times and MS spectra of the candidate analytes in the apple juice with those
of reference compounds, followed by co-chromatography. Varying extraction times (15
- 120 min), desorption time (15 - 120 min), and solvents (acetonitrile, methanol)
revealed that stirring a low sample volume of 20 mL with the PDMS-coated bar for 60
min at room temperature, followed by extractive stir-bar desorption with only 100 µL
acetonitrile for 60 min achieved the reproducible and sensitive detection of the odor-
active esters (5-7, 9, 10, 12, 14) and β-damascenone (3) by means of UHPLC-MS/MS.
After UHPLC separation in less than 10 min, β-damascenone (3) was quantitated using
3-d as internal standard, the esters 5-7, 9, 10, 12, and 14 by using 5-d , 6-d , 7-d , 9-
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d , 10-d , 12-d , 14-d as internal standards, and ester 8 was analyzed via 12-d , ester
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The polyphenols phlorizin (18), epicatechine (20) and chlorogenic acid (21)
were best quantitated by simply injecting the membrane filtered juice into the LC-MS-
system with the use of dihydrorobinetin (22) as internal standard for epicatechine (20)
and phlorizine (19), and caffeic acid ethyl ester-d (23-d ) as internal standard for
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chlorogenic acid (21), Figure 5. Spiking apple juice samples with additional amounts
of the analytes, followed by derivatization and UHPLC-MS/MS analysis revealed a
recovery of the analytes in a range between 78.5 (chlorogenic acid) and 101.8 % found
for methyl 2-methylbutanoate (Table 1). This method allowed the analysis of the
odorants with very low LODs and LOQs, e.g. the LOQ ranged from 0.002 (ethyl
hexanoate) and 0.004 (ß-damascenone) to 0.723 µmol/L found for butyl acetate, while
somewhat higher LOQs of 1.031 (epicatechin) to 2.521 µmol/L (chlorogenic acid) were
found for the polyphenols (Table 1).
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Unified Flavor Quantitation of Apple Juice. The analytical methods
developed were applied for the unified quantitation of key odorants and tastants over
a large dynamic range of up to 6 orders of magnitude in six commercially available
apple juice samples (A-F), e.g. the concentrations of the compounds analyzed ranged
from 0.002 µmol/L for propyl 2-methylbutanoate and 0.01 µmol/L (ß-damascenone) to
89865 µmol/L for glucose (Table 2).
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Glucose and fructose were analyzed using the same MRM-transitions but could
be separated chromatographically. The sugars were found as the quantitatively
predominant compound in concentrations between 46560 (juice F) to 89865 µmol/L
(juice E) for glucose (15) and 342473 (juice E) to 429645 µmol/L (juice D) for fructose
(16). These data are well in line with the levels of 50 - 180 mmol/L (15) and 367 - 533
mmol/L reported earlier.37 The concentrations of malic acid (16) and citric acid (17)
ranged between 25707 (juice E) and 66736 µmol/L (F) and between 214.38 (juice D)
and 485.38 µmol/L (E) and are in the same range as previously published data on
malic acid (19.4 - 45.8 mmol/L) and citric acid (0.067 - 0.515 mmol/L) in apple juice.38
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Compared to glucose and organic acids, relatively low amounts were found for
the taste-active polyphenols, e.g. the sweet tasting phlorizin (19) was present in levels
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between 0.41 (juice C) and 28.68 µmol/L (juice A) and the concentration of the
astringent chlorogenic acid (21) and epicatechin (20) ranged from 44.94 (juice D) to
205.07 µmol/L (juice B) and from 0.40 (juice A) to 26.93 µmol/L (juice B), respectively.
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The odor-active aldehydes E-2-hexenal (1), hexanal (2), and acetaldehyde (4)
were determined in concentration ranges of 0.62 (juice B) – 2.61 µmol/L (juice D), 4.44
(juice D) – 11.9 µmol/L (juice B), and 30.83 (juice A) – 135.34 µmol/L (juice B), while
comparatively low levels of 0.01 - 0.02 µmol/L were found for the cooked apple-like
smelling β-damascenone (3). These data collected by means of UHPLC-MS/MS-SIDA
are well in alignment with previous reports on the quantitation of E-2-hexenal (0.51 –
15.3 µmol/L), hexanal (0.3 – 4.3 µmol/L), acetaldehyde (44 - 551 µmol/L), and ß-
damascenone (0.005 – 0.089 µmol/L) in apple juices using SAFE-distillation for volatile
isolation and HRGC-MS for SIDA-analysis.28,29,39–41 Also the concentrations of the
odor-active esters (5 - 14) with the highest levels of 1.83 (juice A) – 7.16 µmol/L (juice
F) and 0.259 (juice A) – 1.068 µmol/L (juice B) found for butyl acetate (6) and ethyl
butanoate (5) and only trace levels (0.0016 – 0.01 µmol/L) found for propyl 2-
methylbutanoate (13) were well in accordance with literature reports on key odorants
in apple juices analyzed by HRGC-MS-SIDA.39,40,28,26
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Whereas the instrumental analysis of volatile odorants and non-volatile tastants
was traditionally separated and performed by means of high-resolution GC-MS and
LC-MS analysis, respectively, the new approach of “unified flavor quantitation” has
been developed to enable a highly accurate, sensitive high-throughput SIDA analysis
of both, odorants and tastants, on one UHPLC-MS/MS platform without time-
consuming separation of the volatile fraction from non-volatiles, e.g. by means of SAFE
distillation.22 Without the need for injecting several extract dilutions, in addition, the
large dynamic range of the fast-scanning mass spectrometer over 6 orders of
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magnitude enabled the unified flavor quantitation in apple juice to capture volatiles and
non-volatiles within a large concentration range, e.g. propyl 2-methylbutanoate (0.0016
– 0.01 µmol/L), ß-damascenone (0.01 - 0.02 µmol/L), ethyl butanoate (0.259 – 1.068
µmol/L), E-2-hexenal (0.62 - 2.61 µmol/L), phlorizin (0.41 - 28.68 µmol/L), citric acid
(214.38 - 485.38 µmol/L) and glucose (up to 90000 µmol/L).
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As the 3-NPH derivatization takes only 30 min and UHPLC-MS/MS analysis
requires only a low juice sample volume (40 µL) and a short chromatographic
separation time of less than 10 min, this method holds promise for the in parallel, high-
throughput quantitation of flavor-active carbonyls, such as, e.g. acetaldehyde, hexanal,
E-2-hexenal, glucose, and fructose and organic acids like malic acid and citric acid in
foods and beverages. This method is perfectly complemented with the direct and
derivatization-free UHPLC-MS/MS analysis of taste-active polyphenols and odor-
active esters without or after target analyte enrichment through stir-bar sorptive
extraction (SBSE). This “unified flavor quantitation” approach is reported here for the
first time using apple juice as an example. However, it will be applied and validated for
additional food categories in order to investigate its broad applicability. This will support
the transition of today’s labor and time consuming, low-throughput analysis of odorants
and tastants into a new era of unified high-performance quantitation of key flavor
molecules.
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Funding
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The project was funded by the Federal Ministry of Education and Research of Germany
(BMBF) under the grant number 01EA1409A.
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Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
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The authors acknowledge the financial support by the Federal Ministry of Education
and Research of Germany in the framework of ENABLE (01EA1409A).
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SUPPORTING INFORMATION AVAILABLE
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Chemical structures and MRM parameters for mass spectrometric analysis of odor and
taste compounds are available free of charge via the Internet at http://pubs.acs.org.
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by liquid chromatography-tandem mass spectrometry, Analytica chimica acta.
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(35) Frank, O.; Kreissl, J. K.; Daschner, A.; Hofmann, T. Accurate Determination of
Reference Materials and Natural Isolates by Means of Quantitative 1H NMR
Spectroscopy, J. Agric. Food Chem. 2014, 62, pp. 2506–2515.
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(36) Barba, C.; Thomas-Danguin, T.; Guichard, E. Comparison of stir bar sorptive
extraction in the liquid and vapour phases, solvent-assisted flavour evaporation
and headspace solid-phase microextraction for the (non)-targeted analysis of
volatiles in fruit juice, LWT--Food Sci. Technol. 2017, 85, pp. 334–344.
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(37) Karadeniz, F.; Ekşi, A. Sugar composition of apple juices, Eur. Food Res. Technol.
2002, 215, pp. 145–148.
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(38) Anna-Katharina Schmid. Entschlüsselung des nicht-flüchtigen Sensometaboloms
von Äpfeln und Apfelsaft und Identifizierung von süß-modulierenden
Triterpencarbonsäuren, 2016.
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(39) Lehmann, K. Entwicklung eines Aromawert-Indexes zur Bewertung der
Aromaqualität von Apfelsäften auf der Basis von molekularer Sensorik, 2016.
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(40) Steinhaus, M.; Bogen, J.; Schieberle, P. Key aroma compounds in apple juice-
changes during juice concentration. In Developments in Food Science : Flavour
Science; Bredie, W. L.P.; Petersen, M. A., Eds.; Elsevier, 2006, pp. 189–192.
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(41) Fuhrmann, E.; Grosch, W. Character impact odorants of the apple cultivars elstar
and cox orange, Nahrung. 2002, 46, pp. 187–193.
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Table 1. Validation Experiments for Unified Quantitation of Key Odorants and
Tastants in Apple Juice after 3-NPH Derivatization and Stir-Bar Sorptive Extraction
(SBSE), Respectively.
Analyte (no.)
RSDa
recoveryb
LODc
[µmol/L]
0.004
0.03
LOQd
[µmol/L]
0.014
0.068
0.004
0.027
0.306
0.723
0.018
0.017
0.143
0.121
0.017
0.014
0.004
0.002
83.3
[%]
[%]
E-2-hexenal (1)
11.2
13.1
11.2
14.2
8.4
107.2
93.1
hexanal (2)
β-damascenone (3)
acetaldehyde (4)
103.9
86.1
0.001
0.009
0.092
0.217
0.006
0.005
0.006
0.036
0.001
0.005
0.001
0.001
23.7
ethyl butanoate (5)
butyl acetate (6)
98.4
10.2
10.4
9.7
93.7
ethyl 2-methylbutanoate (7)
ethyl 2-methylpropanoate (8)
ethyl propanoate (9)
hexyl acetate (10)
pentyl acetate (11)
100.2
93.6
10.7
8.6
91.7
99.3
12.2
90.9
methyl 2-methylbutanoate (12) 11.2
101.8
90.9
propyl 2-methylbutanoate (13)
ethyl hexanoate (14)
glucose (15)
4.8
8.3
9.6
5.2
4.8
16.9
6.4
9.5
3.9
92.1
96.5
fructose (16)
98.5
21.2
70.9
malic acid (17)
102.4
95.9
0.14
0.37
citric acid (18)
0.05
0.15
phlorizin (19)
100.0
81.7
0.305
0.123
0.479
1.596
1.031
epicatechine (20)
chlorogenic acid (21)
78.5
2.512
a
b
c
d
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Recovery (%), Relative standard deviation (%), Limit of Detection (µmol/L), Limit
of quantitation.
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Table 2. Concentrations of Odorants and Tastants in Commercial Apple Juices.
concentration (µmol/L; std. dev.) in apple juice samples
Analyte (no.)
A
B
C
D
E
F
fructose
403363
377803
379634
429645
342473
384408
(
16)
glucose
15)
malic acid
17)
citric acid
18)
(± 40130) (± 37771) (± 14070) (± 2081) (± 10063) (± 14837)
83315
(± 2963)
42384
(± 1076)
328.71
65131
(± 9021)
40510
(± 911)
477.05
63999
(± 3729)
28019
(± 919)
393.37
67736
(± 6473)
28309
(± 755)
214.38
89865
(± 7864)
25707
(± 1805)
485.38
46560
(± 7582)
66736
(± 4823)
350.05
(
(
(
(± 48.83) (± 136.49) (± 61.29)
(± 36.94) (± 149.53) (± 70.18)
chlorogenic
acid (21)
84.89
(± 0.30)
30.83
205.07
(± 7.76)
135.34
75.24
(± 4.07)
66.82
44.94
(± 1.10)
59.77
48.59
(± 2.81)
91.83
136.10
(± 7.92)
62.18
acetaldehyde
(
4)
phlorizin
19)
epicatechin
20)
hexanal
2)
(± 4.61)
28.68
(± 0.82)
0.49
(± 0.09)
8.38
(± 19.43) (± 19.02) (± 12.21)
(± 6.67)
8.33
(± 0.43)
1.67
(± 0.21)
8.81
(± 13.22)
14.15
(± 0.57)
4.20
(± 0.29)
6.02
14.73
(± 0.21)
26.93
0.41
(± 0.08)
2.42
8.82
(± 0.42)
1.10
(
(
(± 0.92)
11.90
(± 0.09)
7.37
(± 0.14)
4.44
(
(± 4.61)
(± 1.36)
(± 1.46)
(± 0.36)
(± 0.96)
(± 0.43)
butyl
acetate (6)
1.83
(± 0.05)
7.00
(± 0.58)
3.06
(± 0.21)
3.14
(± 0.25)
4.36
(± 0.33)
7.16
(± 0.85)
E-2-hexenal
0.85
(± 0.18)
0.62
(± 0.13)
1.63
(± 0.05)
2.61
(± 0.07)
1.87
(± 0.28)
2.45
(± 0.11)
(1)
ethyl
0.259
1.068
(± 0.105)
1.609
(± 0.128)
0.592
1.014
(± 0.069)
0.646
(± 0.043)
0.264
0.524
(± 0.021)
0.78
(± 0.043)
0.303
0.555
(± 0.086)
0.935
(± 0.084)
0.362
0.657
(± 0.046)
1.397
(± 0.226)
0.506
butanoate (5) (± 0.018)
hexyl
acetate (10)
0.345
(± 0.023)
0.154
ethyl
propanoate (9) (± 0.011)
(± 0.051)
0.181
(± 0.014)
(± 0.030)
0.125
(± 0.021)
(± 0.024)
0.106
(± 0.016)
(± 0.082)
0.108
(± 0.032)
(± 0.036)
0.429
(± 0.048)
pentyl acetate
11)
ethyl 2-methyl
0.058
(± 0.006)
0.096
(
0.368
0.22
0.206
0.17
0.818
butanoate (7) (± 0.009)
methyl 2-methyl
0.016
butanoate (12) (± 0.002)
ethyl
0.016
hexanoate (14) (± 0.002)
ethyl 2-methyl
0.009
(± 0.031)
0.061
(± 0.005)
0.056
(± 0.005)
0.039
(± 0.016)
0.03
(± 0.006)
0.063
(± 0.004)
0.037
(± 0.012)
0.027
(± 0.002)
0.028
(± 0.001)
0.018
(± 0.034)
0.041
(± 0.012)
0.043
(± 0.003)
0.020
(± 0.099)
0.081
(± 0.011)
0.063
(± 0.007)
0.023
propanoate (8) (± 0.001)
(± 0.004)
(± 0.001)
(± 0.001)
(± 0.004)
(± 0.002)
β-damascenone 0.0100
0.0140
0.0134
0.0181
0.0203
0.0150
(
3)
(± 0.0005) (± 0.0005) (± 0.0005) (± 0.0008) (± 0.0007) (± 0.0008)
0.01 0.0016 0.0033 0.003 0.0045
propyl 2-methyl 0.0029
butanoate (13) (± 0.0002) (± 0.0004) (± 0.0001) (± 0.0001) (± 0.0001) (± 0.0003)
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FIGURE LEGEND
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Figure 1.
Dose/threshold plot showing the concentrations of 230 key food
odorants, which by definition exceed their threshold concentration in
5
2
27 food samples analyzed, plotted against their human threshold
concentration. To increase readability, the 230 key food odorants are
grouped in different chemical classes.
Figure 2.
Figure 3.
Derivatization of carbonyl compounds with 3-nitrophenylhydrazine (3-
NPH)
using
N-(3-dimethylaminopropyl)-N’-ethylcarbodiimide
hydrochloride (EDC) as catalyst.
Chemical structures of analyzed odorants and tastants: E-2-hexenal
(1), hexanal (2), β-damascenone (3), acetaldehyde (4), ethyl
butanoate (5), butyl acetate (6), ethyl 2-methylbutanoate (7), ethyl 2-
methylpropanoate (8), ethyl propanoate (9), hexyl acetate (10), pentyl
acetate (11), methyl 2-methylbutanoate (12), propyl 2-
methylbutanaote (13), ethyl hexanoate (14), glucose (15), fructose
(16) malic acid (17), citric acid (18), phlorizin (19), epicatechine (20),
and chlorogenic acid (21).
Figure 4.
UHPLC-MS/MS analysis of an apple juice sample showing the mass
transitions of E-2-hexenal (1), hexanal (2), acetaldehyde (4), glucose
(15), fructose (16), malic acid (17) and citric acid (18), as well as the
internal standards E-2-hexenal-d2 (1-d2), hexanal-d5 (2-d5),
13
13
acetaldehyde-d (4-d ), glucose- C (15- C ), malic acid-d (17-d ),
3
3
6
6
3
3
13
13
and citric acid- C (18- C ) after 3-NPH derivatization. Signal
2
2
intensity of each mass transition is normalized.
Figure 5.
UHPLC-MS/MS analysis of an apple juice sample showing the mass
transitions of β-damascenone (3), ethyl butanoate (5), butyl acetate
(6), ethyl 2-methylbutanoate (7), ethyl 2-methylpropanoate (8), ethyl
propanoate (9), hexyl acetate (10), pentyl acetate (11), methyl 2-
methylbutanoate (12), propyl 2-methylbutanaote (13), ethyl hexanoate
(14), phlorizin (19), epicatechine (20), chlorogenic acid (21) and the
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internal standards β-damascenone-d (3-d ), ethyl butyrate-d (5-d ),
4
4
5
5
butyl-d5 acetate (6-d ), ethyl 2-methyl-d butyrate (7-d ), ethyl
5
3
3
propanoate-d (9-d ), hexyl-d acetate (10-d ), methyl 2-methyl-d
3
5
5
5
5
butyrate (12-d ), ethyl hexanoate-d (14-d ), dihydrorobinetin (22),
3
5
5
and caffeic acid ethyl ester-d (23-d ) after SBSE enrichment (esters)
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5
or direct injection (polyphenols). Signal intensity of each mass
transition is normalised.
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Hofstetter, Dunkel, Hofmann, Figure 1
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Hofstetter, Dunkel, Hofmann, Figure 2
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