Unified Flavor Quantitation: Towards High-Throughput Analysis of Key

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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 Downloaded from pubs.acs.org on July 21, 2019

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Journal of Agricultural and Food Chemistry

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Unified Flavor Quantitation: Towards High-Throughput

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Analysis of Key Food Odorants and Tastants by Means of

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UHPLC-MS/MS

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Christoph Konrad Hofstetter†, Andreas Dunkel#‡ and Thomas Hofmann† #‡

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†Chair

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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

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for Food Systems Biology at the Technical University of Munich,

Lise-Meitner-Str. 34, D-85354 Freising, Germany, and

10 ‡Bavarian

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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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PHONE

+49-8161/71-2902

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FAX

+49-8161/71-2949

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E-MAIL

[email protected]

To whom correspondence should be addressed

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Abstract

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As foods are perceived through combined inputs from taste and odor, which are

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determined by the concentration of the individual odor and taste molecules, the unified

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high-throughput quantitation of volatile odorants and non-volatile tastants with the very

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same instrumental set-up has been a long-standing, but yet unmet dream. The

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research presented here for the first time demonstrates, after only minimal sample

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work-up, the highly accurate, rapid, and sensitive unified quantitation of odorants and

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tastants of key flavour molecules in apple juice on a single UHPLC-MS/MS platform

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over a large dynamic range of up to 6 orders of magnitude. While flavor active

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aldehydes, ketones and organic acids were analyzed after derivatization with 3-

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nitrophenylhydrazine, taste-active polyphenols and odor-active esters were directly

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analyzed by means of UHPLC-MS/MS with and without target analyte enrichment

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through stir-bar sorptive extraction. This “unified flavor quantitation” approach holds

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promise to accelerate the transition of today’s labor and time consuming, low-

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throughput analysis of odorants and tastants into a new era of high-performance

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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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ACS Paragon Plus Environment

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Introduction

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The health and environmental challenges arising from modern lifestyles call for

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avantgardistic scientific approaches helping to better understanding consumers’

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decisions on food choice and targeting food engineering to deliver healthy nutrition

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without compromising aroma and taste and authentic eating experiences with positive

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impact on the environment and food safety. To overcome flavor defects in food

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products, induced by reducing levels of highly palatable ingredients (salt, sugar, fat

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etc.), by adding health-promoting phytometabolites, or by using alternative raw

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materials (plant-based proteins, algae-based lipids etc.), respectively, the consumer

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acceptance of such products depends on new solutions and technologies capable of

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fine-tuning flavor deviations for the delivery of truly authentic flavor signatures. To

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achieve this, a thorough understanding of how our chemical senses olfaction and taste

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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-

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guided identification to the discovery of volatile odorants and non-volatile tastants by

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means of aroma extract dilution analysis1,2 and taste dilution analysis3,4 has

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demonstrated the sheer unlimited variations in food flavors to be created by a

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“combinatorial chemosensory code” of a limited number of key molecules. Food odor

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recognition has been shown to consult a large repertoire of ~380 odorant receptors to

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sense a surprisingly small center group of 3 - 40 key food odorants per food item out

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of a total of ~230 key food odorants among the 10.000 food-born volatiles identified.5

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Complementing the constructive sense of olfaction, a small number of only ~30 taste

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receptors is designed to sense chemically diverse non-volatiles, among which 15 - 40

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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,

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sweet, sour, salty, and umami.6

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Most impressively, minimal chemosensory recombinants of 3 - 40 key odorants

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and 15 - 40 key tastants have been demonstrated to be truly necessary and sufficient

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for constructing the authentic percept of a specific food’s flavor in our brain such as,

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e.g. black tea,7,8 prawns,9,10 and red wine11. Therefore, the key mechanism by which

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the brain encodes perceptual representations of behaviorally relevant food items is

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considered through the synthesis of combinatorial chemosensory inputs into a unique

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perceptual experience (“flavor object”), rather than through individual molecules.12–14

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Within the recent decades, most key odorants and basic taste compounds have been

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already successfully identified and, within a food category, small quantitative rather

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than chemical structure variations in odorant/tastant codes were concluded to trigger

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perceived differences in sensory phenotypes of a food category.5,15 Therefore,

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changes in odor and taste profiles from the raw materials through the various

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manufacturing process intermediates all the way up-stream to the consumer’s plate

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may be monitored by high-precision and high-throughput mass spectrometric profiling

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of the entire population of a given food’s key flavor compounds, coined

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“sensometabolome”.16,17

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In order to counteract the analytical challenges in accurate quantitation of

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odorants and tastants, which are largely differing in concentration, volatility, and

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chemical stability, the so-called stable isotope dilution analysis (SIDA) has been

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successfully introduced in flavor research, using stable isotope (13C, 2H)-labeled

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analogue molecules of the odorants and tastants as most suitable internal standards

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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-

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MS, GC/GC-MS, and GC˟GC-MS, respectively, is highly accurate and sensitive, gas

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chromatographic analysis is limited to volatile analysis and, therefore, usually requires

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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.22 Another challenge in odorant analysis

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is the large concentration range of odorants in food that need to be captured

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analytically. Using the earlier reported meta-analytical data on 230 key food odorants,

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which by definition exceed their threshold concentration in 227 food samples

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analyzed,5 the concentrations of these key food odorants, grouped in different chemical

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classes, are plotted against their human threshold concentration in Figure 1. This plot

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indicates that the key food odorants, depending on their chemical class and individual

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threshold concentration, are present in a sensorially relevant concentration range of

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~1 pmol/kg as found for thiols and sulphides, respectively, all the way up to ~10-100

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mmol/kg as typical for alcohols. In consequence, odorant quantitation by means of GC-

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MS requires that different sample volumes are worked up or that the aroma extract

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prepared is injected several times at different dilutions in order to account for the

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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

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chromatography hyphenated to a mass spectrometer. Sensorially relevant levels of

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taste active compounds, that means by definition concentrations in foods exceeding

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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 terpenoids23 as well

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as taste enhancers,24,16,10,25 and in the mmol/kg range as typically found for basic taste

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compounds such as, e.g. amino acids, organic acids, and carbohydrates,

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respectively.24,16,10,25



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As foods are perceived through combined inputs from taste and odor, which are

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determined by the concentration of the individual odor and taste molecules, the unified

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high-throughput quantitation of volatile odorants and non-volatile tastants with the very

123

same instrumental set-up would be highly desirable. New developments in ultra-high

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performance liquid chromatography (UHPLC) and fast-scanning mass spectrometers

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now hold promise to enable, after minimal sample work-up, the highly accurate and

126

sensitive unified quantitation of odorants and tastants on a single instrument platform

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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

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UHPLC-MS/MS quantitation of key odorants and tastants in foods using apple juice as

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an example. Based on previous research, E-2-hexenal (1), hexanal (2), β-

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damascenone (3), acetaldehyde (4), ethyl butanoate (5), butyl acetate (6), ethyl 2

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methylbutanoate (7), ethyl 2 methylpropanoate (8), ethyl propanoate (9), hexyl acetate

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(10), pentyl acetate (11), methyl 2 methylbutanoate (12), propyl 2-methylbutanaote

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(13), and ethyl hexanoate (14) are considered key odorants in apple juice (Figure 3),26–

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quantitation method should comprise the sweet carbohydrates glucose (15) and

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fructose (16), the sour tasting organic acids malic acid (17) and citric acid (18), the

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sweet tasting polyphenol phlorizin (19), as well as the key astringent polyphenols

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epicatechin (20) and chlorogenic acid (21).30–33

and, therefore, should be analyzed in the following study. In addition, the unified

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Materials and Methods

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Chemicals. The following compounds were obtained commercially: 3-

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nitrophenylhydrazine hydrochloride, N-(3-dimethylaminopropyl)-N’-ethylcarbodiimide

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hydrochloride, acetic acid, malic acid, citric acid, glucose, phlorizine, chlorogenic acid,


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(-)-epicatechine, glucose-13C6, malic acid-d3, citric acid-1,5-13C2, chlorogenic acid ethyl

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ester-d5 (Sigma-Aldrich, Steinheim, Germany), pyridine (Merck, Darmstadt, Germany),

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E-2-hexenal, hexanal, β-damascenone, acetaldehyde, ethyl hexanoate, butyl acetate,

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methyl 2-methylbutanoate, ethyl 2-methylbutanoate, hexyl acetate, ethyl butanoate

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(Givaudan, Vernier, Suisse), dihydrorobinetin (Extrasynthèse, Genay Cedex, France),

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E-2-hexenal-d2, β-damascenone-d4, hexanal-d5, ethyl hexanoate-d5, butyl-d5 acetate,

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methyl 2-methyl-d3 butyrate, ethyl 2-methyl-d3-butyrate, hexyl-d5 acetate, ethyl

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butyrate-d5 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,

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Seelze, Germany). Acetonitrile-d3 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

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water advantage A 10 water system (Millipore, Molsheim, France). Triplicates from four

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clear apple juice samples A, C, D and E, and two cloudy apple juice samples B and F

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were purchased from a local retailor (Freising, Germany).

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Sample Preparation. Apple juice samples were membrane-filtered (Minisart

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RC 15, 0.45 μm; Sartorius AG, Göttingen, Germany) and the following internal

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standard mixture in acetonitrile/water (20/80, v/v) was added before further sample

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work-up: E-2-hexenal-d2 (1-d2, 1200 µg/kg), hexanal-d5 (2-d5, 530 µg/kg), (2E)-β-

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damascenone-d4 (3-d4, 7.4 µg/kg), acetaldehyde-d3 (4-d3, 8000 µg/kg), ethyl butyrate-

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d5 (5-d5, 44 µg/kg), butyl-d5 acetate (6-d5, 840 µg/kg), ethyl 2-methyl-d3 butyrate (7-d3,

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24 µg/kg), ethyl propanoate-d5 (9-d5, 35 µg/kg), hexyl-d5 acetate (10-d5, 175 µg/kg),

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methyl 2-methyl-d3 butyrate (12-d3, 7.2 µg/kg), ethyl hexanoate-d5 (14-d5, 3.4 µg/kg),

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glucose-13C6 (15-13C6, 15.5 g/kg), malic acid-d3 (17-d3, 5.3 g/kg), citric acid-13C2 (18-

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13C

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(23-d5, 78.5 mg/kg), respectively.

2,

63.1 mg/kg), dihydrorobinetin (22, 160.5 mg/kg), and caffeic acid ethyl ester-d5



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Quantitation of Flavor-Active Aldehydes, Ketones, and Organic Acids after

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3-NPH Derivatization. Following a method reported for the analysis of short and

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branched fatty acids,34 aliquots (40 µL) of the filtered apple juice samples spiked with

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the internal standards were mixed with a solution (20 µL) of 3-nitrophenyl hydrazine

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(3-NPH; 200 mmol/L) in acetonitrile/water (50/50, v/v) and a solution (20 µL) of N-(3-

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dimethylaminopropyl)-N’-ethylcarbodiimide (EDC, 120 mmol/L) in acetonitrile/water

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(50/50, v/v) containing 6 % pyridine. After incubation for 30 min at 40 °C, the solutions

177

were made up with acetonitrile/water (50/50, v/v) to 1.0 mL and, then, an aliquot (1 µL)

178

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

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mg/L), hexanal (5.3 mg/L), acetaldehyde (80 mg/L), malic acid (52.9 mg/L), citric acid

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(0.94 mg/L), and glucose (155.6 mg/L) was prepared in acetonitrile/water (50/50, v/v)

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and the exact concentration of each reference compound verified by means of

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quantitative NMR. This stock solution was diluted 1:2, 1:3, 1:5, 1:10, 1:50, 1:100,

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1:1000 with acetonitrile/water (50/50, v/v). To each dilution the same amount of internal

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Standard (IS) was added for an end concentration of 1200 µg/L E-2-hexenal, 530 µg/L

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hexanal, 8 mg/L acetaldehyde, 5.3 g/L malic acid, 15.5 g/L glucose and 94 mg/L citric

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acid. 40 µl of each standard solution were used for derivatization using the instructions

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above. For recovery experiments, the analytes were spiked into an apple juice using

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the concentration ranges of the internal standard calibration curve as triplicates. After

190

addition of internal standard solutions, the samples were prepared as detailed above.

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For the determination of the limit of detection (LOD) and the limit of quantitation (LOQ)

192

the 1:1000 dilution was further diluted 1:10, 1:100 and 1:1000 and the signal to noise

193

ratio was measured using the MultiQuant software (Sciex, Darmstadt, Germany). The

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LOD value was set to a signal to noise ratio of 3 and the LOQ to a signal to noise ratio

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of 10, respectively. ACS Paragon Plus Environment

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UHPLC-MS/MS Analysis of 3-NPH-Derivatives. The samples were separated

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by means of a Nexera UHPLC (Shimadzu Europa GmbH, Duisburg, Germany)

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consisting of two LC pump systems 30AD, a DGU-20A5 degasser, a SIL-30AC

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autosampler, a CTO-30A column oven, and a CBM-20A controller, and equipped with

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a 100 x 2.1 mm, 100 A, Kinetex 1.7 µm C18 column (Phenomenex, Aschaffenburg,

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Germany). A gradient of 0.1 % formic acid in water (solvent A) and 0.1 % formic acid

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in acetonitrile (solvent B) was used (0.4 mL/min): 0 min, 20 % B; 2 min 20 % B; 4 min,

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100 % B; 6 min, 100 % B; 7 min, 20 % B; 10 min, 20 % B. Gradient SBSE: 0 min, 40

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% B; 1.5 min, 40 % B; 4.5 min, 60 % B; 6 min, 100 % B; 7 min, 100 % B; 9 min, 40 %

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B; 10 min, 40 % B. The UHPLC was connected to a QTRAP 6500 mass spectrometer

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(Sciex, Darmstadt, Germany), controlled by the Analyst 1.6.2 software (Sciex), and

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operated in the full-scan mode (ion spray voltage: -4500 V) with the following

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instrument settings: curtain gas (35 V), temperature (450 °C), gas 1 (55 V), gas 2 (65

209

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,

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PDMS-stir bars (10 mm in length, 0.5 mm film thickness; Gerstel GmbH & Co.KG,

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Mühlheim, Germany) were thermally pre-treated for 1 h at 280 °C using the Tube

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Conditioner TC 2 for (Gerstel GmbH & Co.KG). After cooling, the stir bars were placed

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into a glass vial (20 mL), an aliquot (20 mL) of apple juice spiked with the internal

215

standard mixture was added and, then, the glass vials were put on a lab shaker (800

216

rpm) for 60 min at room temperature. Thereafter, the stir bars were washed with

217

deionized water (5 mL), placed into a glass vial (1.5 mL) containing acetonitrile (100

218

µL), followed by shaking (800 rpm) for 60 min. An aliquot (3 µL) of the organic solution

219

was then directly injected into the UHPLC-MS/MS system.



220

Internal Standard Calibration Curve. A stock solution of ethyl hexanoate (34

221

µg/L), butyl acetate (8.4 mg/L), methyl 2-methylbutanoate (72 µg/L), ethyl 2-

222

methylbutanoate (0.24 mg/L), hexyl acetate (1.7 mg/L), ethyl butanoate (0.44 mg/L),

223

and β-damascenone (74 µg/L) was prepared in acetonitrile/water (50/50, v/v). This

224

stock solution was diluted 1:2, 1:3, 1:5, 1:10, 1:50, 1:100, 1:1000 with acetonitrile/water

225

(50/50, v/v). To each dilution the same amount of internal standard was added to reach

226

an end concentration of 3.4 µg/L for ethyl hexanoate, 840 µg/L for butyl acetate, 7.2

227

µg/L for methyl 2-methylbutanoate, 24 µg/L for ethyl 2-methylbutanoate, 175 µg/L for

228

hexyl acetate, 44 µg/L for ethyl butanoate, and 7.4 µg/L for β-damascenone,

229

respectively. Aliquots (20 mL) of each standard solution were used for SBSE as

230

detailed above. For recovery experiments, the analytes were spiked into an apple juice

231

using the concentration ranges of the internal standard calibration curve as triplicates.

232

After addition of the internal standards, the samples were prepared as reported above.

233

For the determination of the LOD and LOQ values the 1:1000 dilution was further

234

diluted 1:10, 1:100 and 1:1000 and the signal to noise ratio was measured using

235

MultiQuant software (Sciex, Darmstadt, Germany). The LOD value was set to a signal

236

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

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samples obtained after SBSE treatment were separated by means of an ExionLC

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(Sciex, Darmstadt, Germany), consisting of two LC pump systems ExionLC AD Pump,

240

an ExionLC degasser, an ExionLC AD autosampler, an ExionLC AC column oven, and

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an ExionLC controller, and equipped with a 100 x 2.1 mm, 1.7 µm, 100 A Kinetex C18

242

column (Phenomenex, Aschaffenburg, Germany). A gradient of 0.1 % formic acid in

243

water (Solvent A) and 0.1 % formic acid in acetonitrile (solvent B) was used (0.4

244

mL/min): 0 min, 40 % B; 1.5 min, 40 % B; 4.5 min, 60 % B; 6 min, 100 % B; 7 min, 100

245

% B; 9 min, 40 % B; 10 min, 40 % B. The UHPLC was hyphenated with a QTRAP ACS Paragon Plus Environment

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6500+ mass spectrometer (Sciex, Darmstadt, Germany), controlled by the Analyst

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1.6.3 software (Sciex), and operated in the full-scan mode (ion spray voltage: 5500 V)

248

using the following instrument settings: curtain gas (35 V), temperature (450 °C), gas

249

1 (55 V), gas 2 (65 V), collision activated dissociation (2 V), and entrance potential (10

250

V). Data interpretation was performed by using MultiQuant software, Analyst 1.6.3

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(Sciex, Darmstadt, Germany) and Microsoft Excel 2016.

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Quantitation of Polyphenols. Aliquots (1 µL) of the membrane-filtered apple

253

juice samples spiked with the added internal standards were directly injected into the

254

UHPLC-MS/MS system.

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Internal Standard Calibration Curve. A stock solution of phlorizin (175 mg/L),

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chlorogenic acid (223 mg/L), and epicatechine (201 mg/L) was prepared in

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acetonitrile/water (20/80, v/v). The exact concentration was verified by means of

258

qNMR. This stock solution was diluted 1:2, 1:5, 1:10, 1:20, 1:50, 1:100, 1:200, 1:500,

259

1:1000 with acetonitrile/water (20/80, v/v). Dihydrorobinetin (160 mg/L) was used as

260

the internal standard for the quantitation of epicatechine and phlorizin, caffeic acid ethyl

261

ester-d5 (171 mg/L; 22-d5) was used for quantitative analysis of chlorogenic acid. For

262

recovery experiments, the analytes were spiked into an apple juice using the

263

concentration ranges of the internal standard calibration curve as triplicates. LC-

264

MS/MS analysis of polyphenols was performed using the parameters described above

265

for 3-NPH-derivatives. For the determination of LOD and LOQ values the 1:1000

266

dilution was further diluted 1:10, 1:100 and 1:1000 and the signal to noise ratio was

267

measured using MultiQuant software (Sciex, Darmstadt, Germany). The LOD value

268

was set to a signal to noise ratio of 3 and the LOQ to a signal to noise ratio of 10,

269

respectively.



270

Quantitative

1H

Nuclear Magnetic Resonance Spectroscopy (qNMR).

271

qNMR was recorded on a 400 MHz Avance III spectrometer (Bruker, Rheinstetten,

272

Germany) equipped with a Broadband Observe BBFO plus Probe (Bruker,

273

Rheinstetten, Germany). Acetonitrile-d3 (600 µL) was used as solvent and chemical

274

shifts are reported in parts per million relative to the acetonitrile-d3 solvent signal. Data

275

processing was performed by using Topspin NMR software vers. 3.2 (Bruker,

276

Eheinstetten, Germany). Quantitative NMR spectroscopy (q-NMR) was performed as

277

reported earlier through calibration of the spectrometer by applying the ERETIC 2 tool

278

using the PULCON methodology.35

279 280

Results and Discussion

281

In order to develop a new analytical approach for high-throughput and joint quantitation

282

of key odorants and tastants in apple juices, the odorants E-2-hexenal (1), hexanal (2),

283

β-damascenone (3), acetaldehyde (4), ethyl butanoate (5), butyl acetate (6), ethyl 2-

284

methylbutanoate (7), ethyl 2-methylpropanoate (8), ethyl propanoate (9), hexyl acetate

285

(10), pentyl acetate (11), methyl 2-methylbutanoate (12), propyl 2-methylbutanaote

286

(13), and ethyl hexanoate (14), as well as the taste-active compounds glucose (15),

287

fructose (16), malic acid (17), citric acid (18), phlorizin (19), epicatechine (20), and

288

chlorogenic acid (21) should be analyzed by means of a single UHPLC-MS/MS

289

plattform.

290

Method Development and Validation Experiments. To enable a highly

291

sensitive MS-detection of flavor-active aldehydes and organic acids over a large

292

dynamic range and after only minimum sample work-up, samples of an apple juice was

293

derivatized upon incubation with 3-nitrophenyl hydrazine (3-NPH) for 15 to 120 min

294

and at temperatures between 20 and 60 °C in the presence of N-(3ACS Paragon Plus Environment

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dimethylaminopropyl)-N’-ethylcarbodiimide in acetonitrile/water and, then, directly

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analyzed by means of UHPLC-MS/MS. The odorants E-2-hexenal (1), hexanal (2) and

297

acetaldehyde (4), as well as the taste compounds glucose (15), fructose (16), malic

298

acid (17), and citric acid (18) were detected and their identity confirmed by comparing

299

the retention times and MS spectra of the candidate analytes in the apple juice with

300

those of reference compounds, followed by co-chromatography. To optimize MS/MS

301

detection for high-sensitivity quantitation, standard solutions of these analytes were

302

derivatized, syringe-infused into the MS ion source, and software-assisted ramping of

303

ion source and ion path parameters in the negative electrospray ionization mode was

304

performed to maximize fragment ion abundance. Varying derivatization time (15 – 120

305

min) and temperature (20 – 60 °C) as well as sample volume (20 - 1000 µL) of apple

306

juice, followed by UHPLC-MS/MS analysis revealed that already the use of a low

307

sample volume of 40 µL and the gentle derivatization conditions (30 min, 40 °C) are

308

fully adequate for unequivocal detection and quantitation of E-2-hexenal (1), hexanal

309

(2), acetaldehyde (4), glucose (15), fructose (16), malic acid (17), and citric acid (18)

310

in apple juice. Quantitation of the analytes was performed in less than 10 min by means

311

of a stable isotope dilution analysis (SIDA) using 1-d2, 2-d5, 4-d3, 15-13C6, 17-d3, and

312

18-13C2 as internal standards spiked to the apple juice samples prior to 3-NPH

313

derivatization (Figure 4). Spiking apple juice samples with additional amounts of the

314

analytes, followed by derivatization and UHPLC-MS/MS analysis revealed good

315

recovery rates for the analytes ranging between 86 and 107 % (Table 1). The limit of

316

detection (LOD) and limit of quantitation (LOQ) were very low for the hydrophobic

317

odorants and ranged from 0.004 and 0.014 µmol/L for E-2-hexenal to 0.03 and 0.068

318

µmol/L for hexanal. In comparison, higher LOD and LOQ were found for the polar taste

319

compounds ranging from 0.05 and 0.15 µmol/L for citric acid to 23.7 and 83.3 µmol/L

320

for glucose (Table 1). ACS Paragon Plus Environment


321

Due to their low MS-sensitivity, a direct UHPLC-MS/MS analysis of the odor-

322

active esters were not possible without any enrichment step. Based on the suitability

323

of stir bar sorptive extraction (SBSE) for the enrichment of hydrophobic odorants prior

324

to HRGC-MS analysis,36 polydimethylsiloxane (PDMS) coated stir bars were added

325

into apple juice samples and, after incubation (15 – 120 min) at room temperature, the

326

stir bars were washed with deionized water and, then, the bound odorants extracted

327

with acetonitrile, followed by UHPLC-MS/MS analysis. The odor-active esters ethyl

328

butanoate (5), butyl acetate (6), ethyl 2-methylbutanoate (7), ethyl 2-methylpropanoate

329

(8), ethyl propanoate (9), hexyl acetate (10), pentyl acetate (11), methyl 2-

330

methylbutanoate (12), propyl 2-methylbutanaote (13), and ethyl hexanoate (14) as well

331

as β-damascenone (3) were detected and their identity confirmed by comparing the

332

retention times and MS spectra of the candidate analytes in the apple juice with those

333

of reference compounds, followed by co-chromatography. Varying extraction times (15

334

- 120 min), desorption time (15 - 120 min), and solvents (acetonitrile, methanol)

335

revealed that stirring a low sample volume of 20 mL with the PDMS-coated bar for 60

336

min at room temperature, followed by extractive stir-bar desorption with only 100 µL

337

acetonitrile for 60 min achieved the reproducible and sensitive detection of the odor-

338

active esters (5-7, 9, 10, 12, 14) and β-damascenone (3) by means of UHPLC-MS/MS.

339

After UHPLC separation in less than 10 min, β-damascenone (3) was quantitated using

340

3-d4 as internal standard, the esters 5-7, 9, 10, 12, and 14 by using 5-d5, 6-d5, 7-d3, 9-

341

d5, 10-d5, 12-d4, 14-d5 as internal standards, and ester 8 was analyzed via 12-d4, ester

342

11 via 7-d3, ester 13 via 10-d5.

343

The polyphenols phlorizin (18), epicatechine (20) and chlorogenic acid (21)

344

were best quantitated by simply injecting the membrane filtered juice into the LC-MS-

345

system with the use of dihydrorobinetin (22) as internal standard for epicatechine (20)

346

and phlorizine (19), and caffeic acid ethyl ester-d5 (23-d5) as internal standard for ACS Paragon Plus Environment

Page 14 of 33

Page 15 of 33


347

chlorogenic acid (21), Figure 5. Spiking apple juice samples with additional amounts

348

of the analytes, followed by derivatization and UHPLC-MS/MS analysis revealed a

349

recovery of the analytes in a range between 78.5 (chlorogenic acid) and 101.8 % found

350

for methyl 2-methylbutanoate (Table 1). This method allowed the analysis of the

351

odorants with very low LODs and LOQs, e.g. the LOQ ranged from 0.002 (ethyl

352

hexanoate) and 0.004 (ß-damascenone) to 0.723 µmol/L found for butyl acetate, while

353

somewhat higher LOQs of 1.031 (epicatechin) to 2.521 µmol/L (chlorogenic acid) were

354

found for the polyphenols (Table 1).

355

Unified Flavor Quantitation of Apple Juice. The analytical methods

356

developed were applied for the unified quantitation of key odorants and tastants over

357

a large dynamic range of up to 6 orders of magnitude in six commercially available

358

apple juice samples (A-F), e.g. the concentrations of the compounds analyzed ranged

359

from 0.002 µmol/L for propyl 2-methylbutanoate and 0.01 µmol/L (ß-damascenone) to

360

89865 µmol/L for glucose (Table 2).

361

Glucose and fructose were analyzed using the same MRM-transitions but could

362

be separated chromatographically. The sugars were found as the quantitatively

363

predominant compound in concentrations between 46560 (juice F) to 89865 µmol/L

364

(juice E) for glucose (15) and 342473 (juice E) to 429645 µmol/L (juice D) for fructose

365

(16). These data are well in line with the levels of 50 - 180 mmol/L (15) and 367 - 533

366

mmol/L reported earlier.37 The concentrations of malic acid (16) and citric acid (17)

367

ranged between 25707 (juice E) and 66736 µmol/L (F) and between 214.38 (juice D)

368

and 485.38 µmol/L (E) and are in the same range as previously published data on

369

malic acid (19.4 - 45.8 mmol/L) and citric acid (0.067 - 0.515 mmol/L) in apple juice.38

370

Compared to glucose and organic acids, relatively low amounts were found for

371

the taste-active polyphenols, e.g. the sweet tasting phlorizin (19) was present in levels ACS Paragon Plus Environment


372

between 0.41 (juice C) and 28.68 µmol/L (juice A) and the concentration of the

373

astringent chlorogenic acid (21) and epicatechin (20) ranged from 44.94 (juice D) to

374

205.07 µmol/L (juice B) and from 0.40 (juice A) to 26.93 µmol/L (juice B), respectively.

375

The odor-active aldehydes E-2-hexenal (1), hexanal (2), and acetaldehyde (4)

376

were determined in concentration ranges of 0.62 (juice B) – 2.61 µmol/L (juice D), 4.44

377

(juice D) – 11.9 µmol/L (juice B), and 30.83 (juice A) – 135.34 µmol/L (juice B), while

378

comparatively low levels of 0.01 - 0.02 µmol/L were found for the cooked apple-like

379

smelling β-damascenone (3). These data collected by means of UHPLC-MS/MS-SIDA

380

are well in alignment with previous reports on the quantitation of E-2-hexenal (0.51 –

381

15.3 µmol/L), hexanal (0.3 – 4.3 µmol/L), acetaldehyde (44 - 551 µmol/L), and ß-

382

damascenone (0.005 – 0.089 µmol/L) in apple juices using SAFE-distillation for volatile

383

isolation and HRGC-MS for SIDA-analysis.28,29,39–41 Also the concentrations of the

384

odor-active esters (5 - 14) with the highest levels of 1.83 (juice A) – 7.16 µmol/L (juice

385

F) and 0.259 (juice A) – 1.068 µmol/L (juice B) found for butyl acetate (6) and ethyl

386

butanoate (5) and only trace levels (0.0016 – 0.01 µmol/L) found for propyl 2-

387

methylbutanoate (13) were well in accordance with literature reports on key odorants

388

in apple juices analyzed by HRGC-MS-SIDA.39,40,28,26

389

Whereas the instrumental analysis of volatile odorants and non-volatile tastants

390

was traditionally separated and performed by means of high-resolution GC-MS and

391

LC-MS analysis, respectively, the new approach of “unified flavor quantitation” has

392

been developed to enable a highly accurate, sensitive high-throughput SIDA analysis

393

of both, odorants and tastants, on one UHPLC-MS/MS platform without time-

394

consuming separation of the volatile fraction from non-volatiles, e.g. by means of SAFE

395

distillation.22 Without the need for injecting several extract dilutions, in addition, the

396

large dynamic range of the fast-scanning mass spectrometer over 6 orders of


Page 16 of 33

Page 17 of 33


397

magnitude enabled the unified flavor quantitation in apple juice to capture volatiles and

398

non-volatiles within a large concentration range, e.g. propyl 2-methylbutanoate (0.0016

399

– 0.01 µmol/L), ß-damascenone (0.01 - 0.02 µmol/L), ethyl butanoate (0.259 – 1.068

400

µmol/L), E-2-hexenal (0.62 - 2.61 µmol/L), phlorizin (0.41 - 28.68 µmol/L), citric acid

401

(214.38 - 485.38 µmol/L) and glucose (up to 90000 µmol/L).

402

As the 3-NPH derivatization takes only 30 min and UHPLC-MS/MS analysis

403

requires only a low juice sample volume (40 µL) and a short chromatographic

404

separation time of less than 10 min, this method holds promise for the in parallel, high-

405

throughput quantitation of flavor-active carbonyls, such as, e.g. acetaldehyde, hexanal,

406

E-2-hexenal, glucose, and fructose and organic acids like malic acid and citric acid in

407

foods and beverages. This method is perfectly complemented with the direct and

408

derivatization-free UHPLC-MS/MS analysis of taste-active polyphenols and odor-

409

active esters without or after target analyte enrichment through stir-bar sorptive

410

extraction (SBSE). This “unified flavor quantitation” approach is reported here for the

411

first time using apple juice as an example. However, it will be applied and validated for

412

additional food categories in order to investigate its broad applicability. This will support

413

the transition of today’s labor and time consuming, low-throughput analysis of odorants

414

and tastants into a new era of unified high-performance quantitation of key flavor

415

molecules.

416 417

Funding

418

The project was funded by the Federal Ministry of Education and Research of Germany

419

(BMBF) under the grant number 01EA1409A.

420



421 422

Notes

423

The authors declare no competing financial interest.

424 425

ACKNOWLEDGMENTS

426

The authors acknowledge the financial support by the Federal Ministry of Education

427

and Research of Germany in the framework of ENABLE (01EA1409A).

428 429

SUPPORTING INFORMATION AVAILABLE

430

Chemical structures and MRM parameters for mass spectrometric analysis of odor and

431

taste compounds are available free of charge via the Internet at http://pubs.acs.org.

432 433 434

LITERATURE CITED

435 436 437

(1) Grosch, W. Evaluation of the Key Odorants of Foods by Dilution Experiments, Aroma Models and Omission, Chem Senses. 2001, 26, pp. 533–545.

438

(2) Schieberle, P. Chapter 17 - New Developments in Methods for Analysis of Volatile

439

Flavor Compounds and their Precursors. In Characterization of Food; Gaonkar, A.

440

G., Ed.; Elsevier Science B.V: Amsterdam, 1995, pp. 403–431.

441

(3) Frank, O.; Ottinger, H.; Hofmann, T. Characterization of an Intense Bitter-Tasting

442

1H,4H-Quinolizinium-7-olate by Application of the Taste Dilution Analysis, a Novel


Page 18 of 33

Page 19 of 33


443

Bioassay for the Screening and Identification of Taste-Active Compounds in

444

Foods, J. Agric. Food Chem. 2001, 49, pp. 231–238.

445

(4) Singldinger, B.; Dunkel, A.; Bahmann, D.; Bahmann, C.; Kadow, D.; Bisping, B.;

446

Hofmann, T. New Taste-Active 3-(O-β-d-Glucosyl)-2-oxoindole-3-acetic Acids and

447

Diarylheptanoids in Cimiciato-Infected Hazelnuts, J. Agric. Food Chem. 2018, 66,

448

pp. 4660–4673.

449

(5) Dunkel, A.; Steinhaus, M.; Kotthoff, M.; Nowak, B.; Krautwurst, D.; Schieberle, P.;

450

Hofmann, T. Nature’s Chemical Signatures in Human Olfaction. A Foodborne

451

Perspective for Future Biotechnology, Angew. Chem. Int. Ed. 2014, 53, pp. 7124–

452

7143.

453

(6) Behrens, M.; Meyerhof, W.; Hellfritsch, C.; Hofmann, T. Sweet and Umami Taste.

454

Natural Products, Their Chemosensory Targets, and Beyond, Angew. Chem. Int.

455

Ed. 2011, 50, pp. 2220–2242.

456

(7) Schuh, C.; Schieberle, P. Characterization of the Key Aroma Compounds in the

457

Beverage Prepared from Darjeeling Black Tea. Quantitative Differences between

458

Tea Leaves and Infusion, J. Agric. Food Chem. 2006, 54, pp. 916–924.

459

(8) Scharbert, S.; Hofmann, T. Molecular Definition of Black Tea Taste by Means of

460

Quantitative Studies, Taste Reconstitution, and Omission Experiments, J. Agric.

461

Food Chem. 2005, 53, pp. 5377–5384.

462

(9) Mall, V.; Schieberle, P. Evaluation of Key Aroma Compounds in Processed Prawns

463

(Whiteleg Shrimp) by Quantitation and Aroma Recombination Experiments, J.

464

Agric. Food Chem. 2017, 65, pp. 2776–2783.

465

(10) Meyer, S.; Dunkel, A.; Hofmann, T. Sensomics-Assisted Elucidation of the Tastant

466

Code of Cooked Crustaceans and Taste Reconstruction Experiments, J. Agric.

467

Food Chem. 2016, 64, pp. 1164–1175.



468

(11) Frank, S.; Wollmann, N.; Schieberle, P.; Hofmann, T. Reconstitution of the Flavor

469

Signature of Dornfelder Red Wine on the Basis of the Natural Concentrations of

470

Its Key Aroma and Taste Compounds, J. Agric. Food Chem. 2011, 59, pp. 8866–

471

8874.

472 473

(12) Jinks, A.; Laing, D. G. The analysis of odor mixtures by humans. Evidence for a configurational process, Physiology & Behavior. 2001, 72, pp. 51–63.

474

(13) Derby, C. D.; Hutson, M.; Livermore, B. A.; Lynn, W. H. Generalization among

475

related complex odorant mixtures and their components. Analysis of olfactory

476

perception in the spiny lobster, Physiology & Behavior. 1996, 60, pp. 87–95.

477

(14) Barkat, S.; Le Berre, E.; Coureaud, G.; Sicard, G.; Thomas-Danguin, T. Perceptual

478

Blending in Odor Mixtures Depends on the Nature of Odorants and Human

479

Olfactory Expertise, Chem Senses. 2012, 37, pp. 159–166.

480

(15) Hofmann, T.; Schieberle, P. Elucidation of the chemosensory code of foods by

481

means of a sensomics approach. In Flavour Science - Proceedings of the XIV

482

Weurman Flavour Research Symposium; Taylor, A. J.; Mottram, D.S., Eds.;

483

Context Products, 2015, p 3-12.

484

(16) Glabasnia, A.; Dunkel, A.; Frank, O.; Hofmann, T. Decoding the Nonvolatile

485

Sensometabolome of Orange Juice (Citrus sinensis), J. Agric. Food Chem. 2018,

486

66, pp. 2354–2369.

487

(17) Hufnagel, J. C.; Hofmann, T. Quantitative Reconstruction of the Nonvolatile

488

Sensometabolome of a Red Wine, J. Agric. Food Chem. 2008, 56, pp. 9190–9199.

489

(18) Schieberle, P.; Hofmann, T. Evaluation of the Character Impact Odorants in Fresh

490

Strawberry Juice by Quantitative Measurements and Sensory Studies on Model

491

Mixtures, J. Agric. Food Chem. 1997, 45, pp. 227–232.


Page 20 of 33

Page 21 of 33


492

(19) Schieberle, P.; Grosch, W. Quantitative analysis of aroma compounds in wheat

493

and rye bread crusts using a stable isotope dilution assay, J. Agric. Food Chem.

494

1987, 35, pp. 252–257.

495

(20) Guth, H.; Grosch, W. Deterioration of soya-bean oil. Quantification of primary

496

flavour compounds using a stable isotope dilution assay, Lebensmittel-

497

Wissenschaft und-Technologie. 1990, 23, pp. 513–522.

498

(21) Stark, T.; Justus, H.; Hofmann, T. Quantitative Analysis of N-Phenylpropenoyl-l-

499

amino Acids in Roasted Coffee and Cocoa Powder by Means of a Stable Isotope

500

Dilution Assay, J. Agric. Food Chem. 2006, 54, pp. 2859–2867.

501

(22) Engel, W.; Bahr, W.; Schieberle, P. Solvent assisted flavour evaporation – a new

502

and versatile technique for the careful and direct isolation of aroma compounds

503

from complex food matrices, European Food Research and Technology. 1999,

504

209, pp. 237–241.

505

(23) Haseleu, G.; Intelmann, D.; Hofmann, T. Identification and RP-HPLC-ESI-MS/MS

506

Quantitation of Bitter-Tasting β-Acid Transformation Products in Beer, J. Agric.

507

Food Chem. 2009, 57, pp. 7480–7489.

508

(24) Mittermeier, V. K.; Dunkel, A.; Hofmann, T. Discovery of taste modulating

509

octadecadien-12-ynoic acids in golden chanterelles (Cantharellus cibarius), Food

510

chemistry. 2018, 269, pp. 53–62.

511

(25) Hillmann, H.; Behr, J.; Ehrmann, M. A.; Vogel, R. F.; Hofmann, T. Formation of

512

Kokumi-Enhancing γ-Glutamyl Dipeptides in Parmesan Cheese by Means of γ-

513

Glutamyltransferase Activity and Stable Isotope Double-Labeling Studies, J. Agric.

514

Food Chem. 2016, 64, pp. 1784–1793.



515

(26) Poll, L. The effect of pulp holding time on the volatile components in apple juice

516

(with and without pectolytic enzyme treatment), LWT--Food Sci. Technol. 1988,

517

21, pp. 87–91.

518

(27) Young, H.; Gilbert, J. M.; Murray, S. H.; Ball, R. D. Causal effects of aroma

519

compounds on royal Gala apple flavors, J. Sci. Food Agric. 1996, 71, pp. 329–336.

520

(28) Steinhaus, M.; Bogen, J.; Schieberle, P., Eds. From apple to juice - changes in

521

key aroma compounds during processing; Deutsche Forschungsanstalt fuer

522

Lebensmittelchemie, 2007.

523

(29) Steinhaus, M.; Baer, S.; Schieberle, P., Eds. Sensory evaluation of commercial

524

apple juices and relation to selected key aroma compounds; Zuercher Hochschule

525

fuer Angewandte Wissenschaften, Institut fuer Chemie und Biologische Chemie,

526

2010.

527

(30) van der Sluis, A. A.; Dekker, M.; Jager, A. de; Jongen, W. M. F. Activity and

528

Concentration of Polyphenolic Antioxidants in Apple. Effect of Cultivar, Harvest

529

Year, and Storage Conditions, J. Agric. Food Chem. 2001, 49, pp. 3606–3613.

530

(31) Oszmianski, J.; Wolniak, M.; Wojdylo, A.; Wawer, I. Comparative study of

531

polyphenolic content and antiradical activity of cloudy and clear apple juices, J.

532

Sci. Food Agric. 2007, 87, pp. 573–579.

533

(32) Fromm, M.; Bayha, S.; Carle, R.; Kammerer, D. R. Characterization and

534

quantitation of low and high molecular weight phenolic compounds in apple seeds,

535

J. Agric. Food Chem. 2012, 60, pp. 1232–1242.

536

(33) Oleszek, W.; Lee, C. Y.; Jaworski, A. W.; Price, K. R. Identification of some

537

phenolic compounds in apples, J. Agric. Food Chem. 1988, 36, pp. 430–432.

538

(34) Han, J.; Lin, K.; Sequeira, C.; Borchers, C. H. An isotope-labeled chemical

539

derivatization method for the quantitation of short-chain fatty acids in human feces ACS Paragon Plus Environment

Page 22 of 33

Page 23 of 33


540

by liquid chromatography-tandem mass spectrometry, Analytica chimica acta.

541

2015, 854, pp. 86–94.

542

(35) Frank, O.; Kreissl, J. K.; Daschner, A.; Hofmann, T. Accurate Determination of

543

Reference Materials and Natural Isolates by Means of Quantitative 1H NMR

544

Spectroscopy, J. Agric. Food Chem. 2014, 62, pp. 2506–2515.

545

(36) Barba, C.; Thomas-Danguin, T.; Guichard, E. Comparison of stir bar sorptive

546

extraction in the liquid and vapour phases, solvent-assisted flavour evaporation

547

and headspace solid-phase microextraction for the (non)-targeted analysis of

548

volatiles in fruit juice, LWT--Food Sci. Technol. 2017, 85, pp. 334–344.

549 550 551

(37) Karadeniz, F.; Ekşi, A. Sugar composition of apple juices, Eur. Food Res. Technol. 2002, 215, pp. 145–148. (38) Anna-Katharina Schmid. Entschlüsselung des nicht-flüchtigen Sensometaboloms

552

von

Äpfeln

und

Apfelsaft

553

Triterpencarbonsäuren, 2016.

und

Identifizierung

von

süß-modulierenden

554

(39) Lehmann, K. Entwicklung eines Aromawert-Indexes zur Bewertung der

555

Aromaqualität von Apfelsäften auf der Basis von molekularer Sensorik, 2016.

556

(40) Steinhaus, M.; Bogen, J.; Schieberle, P. Key aroma compounds in apple juice-

557

changes during juice concentration. In Developments in Food Science : Flavour

558

Science; Bredie, W. L.P.; Petersen, M. A., Eds.; Elsevier, 2006, pp. 189–192.

559

(41) Fuhrmann, E.; Grosch, W. Character impact odorants of the apple cultivars elstar

560

and cox orange, Nahrung. 2002, 46, pp. 187–193.

561 562



563 564 565

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. RSDa

recoveryb

LODc

LOQd

[%]

[%]

[µmol/L]

[µmol/L]

E-2-hexenal (1)

11.2

107.2

0.004

0.014

hexanal (2)

13.1

93.1

0.03

0.068

β-damascenone (3)

11.2

103.9

0.001

0.004

acetaldehyde (4)

14.2

86.1

0.009

0.027

8.4

98.4

0.092

0.306

butyl acetate (6)

10.2

93.7

0.217

0.723

ethyl 2-methylbutanoate (7)

10.4

100.2

0.006

0.018

9.7

93.6

0.005

0.017

10.7

91.7

0.006

0.143

hexyl acetate (10)

8.6

99.3

0.036

0.121

pentyl acetate (11)

12.2

90.9

0.001

0.017

methyl 2-methylbutanoate (12)

11.2

101.8

0.005

0.014

propyl 2-methylbutanoate (13)

4.8

90.9

0.001

0.004

ethyl hexanoate (14)

8.3

92.1

0.001

0.002

glucose (15)

9.6

96.5

23.7

83.3

fructose (16)

5.2

98.5

21.2

70.9

malic acid (17)

4.8

102.4

0.14

0.37

citric acid (18)

16.9

95.9

0.05

0.15

phlorizin (19)

6.4

100.0

0.305

1.596

epicatechine (20)

9.5

81.7

0.123

1.031

chlorogenic acid (21)

3.9

78.5

0.479

2.512

Analyte (no.)

ethyl butanoate (5)

ethyl 2-methylpropanoate (8) ethyl propanoate (9)

566 567

Page 24 of 33

a Recovery

(%), b Relative standard deviation (%), c Limit of Detection (µmol/L), d Limit of quantitation.

568 569 570 ACS Paragon Plus Environment

Page 25 of 33


571 572

Table 2. Concentrations of Odorants and Tastants in Commercial Apple Juices. Analyte (no.) fructose (16) glucose (15) malic acid (17) citric acid (18) chlorogenic acid (21) acetaldehyde (4) phlorizin (19) epicatechin (20) hexanal (2) butyl acetate (6) E-2-hexenal (1) ethyl butanoate (5) hexyl acetate (10) ethyl propanoate (9) pentyl acetate (11) ethyl 2-methyl butanoate (7) methyl 2-methyl butanoate (12) ethyl hexanoate (14) ethyl 2-methyl propanoate (8) β-damascenone (3) propyl 2-methyl butanoate (13)

573 574

concentration (µmol/L; std. dev.) in apple juice samples A 403363 (± 40130) 83315 (± 2963) 42384 (± 1076) 328.71 (± 48.83) 84.89 (± 0.30) 30.83 (± 4.61) 28.68 (± 0.82) 0.49 (± 0.09) 8.38 (± 4.61) 1.83 (± 0.05) 0.85

B 377803 (± 37771) 65131 (± 9021) 40510 (± 911) 477.05 (± 136.49) 205.07 (± 7.76) 135.34 (± 19.43) 14.73 (± 0.21) 26.93 (± 0.92) 11.90 (± 1.36) 7.00 (± 0.58) 0.62

C 379634 (± 14070) 63999 (± 3729) 28019 (± 919) 393.37 (± 61.29) 75.24 (± 4.07) 66.82 (± 19.02) 0.41 (± 0.08) 2.42 (± 0.09) 7.37 (± 1.46) 3.06 (± 0.21) 1.63

D 429645 (± 2081) 67736 (± 6473) 28309 (± 755) 214.38 (± 36.94) 44.94 (± 1.10) 59.77 (± 12.21) 8.82 (± 0.42) 1.10 (± 0.14) 4.44 (± 0.36) 3.14 (± 0.25) 2.61

E 342473 (± 10063) 89865 (± 7864) 25707 (± 1805) 485.38 (± 149.53) 48.59 (± 2.81) 91.83 (± 6.67) 8.33 (± 0.43) 1.67 (± 0.21) 8.81 (± 0.96) 4.36 (± 0.33) 1.87

F 384408 (± 14837) 46560 (± 7582) 66736 (± 4823) 350.05 (± 70.18) 136.10 (± 7.92) 62.18 (± 13.22) 14.15 (± 0.57) 4.20 (± 0.29) 6.02 (± 0.43) 7.16 (± 0.85) 2.45

(± 0.18)

(± 0.13)

(± 0.05)

(± 0.07)

(± 0.28)

(± 0.11)

0.259 (± 0.018) 0.345 (± 0.023) 0.154 (± 0.011) 0.058 (± 0.006) 0.096 (± 0.009) 0.016 (± 0.002) 0.016 (± 0.002) 0.009 (± 0.001) 0.0100 (± 0.0005) 0.0029 (± 0.0002)

1.068 (± 0.105) 1.609 (± 0.128) 0.592 (± 0.051) 0.181 (± 0.014) 0.368 (± 0.031) 0.061 (± 0.005) 0.056 (± 0.005) 0.039 (± 0.004) 0.0140 (± 0.0005) 0.01 (± 0.0004)

1.014 (± 0.069) 0.646 (± 0.043) 0.264 (± 0.030) 0.125 (± 0.021) 0.22 (± 0.016) 0.03 (± 0.006) 0.063 (± 0.004) 0.037 (± 0.001) 0.0134 (± 0.0005) 0.0016 (± 0.0001)

0.524 (± 0.021) 0.78 (± 0.043) 0.303 (± 0.024) 0.106 (± 0.016) 0.206 (± 0.012) 0.027 (± 0.002) 0.028 (± 0.001) 0.018 (± 0.001) 0.0181 (± 0.0008) 0.0033 (± 0.0001)

0.555 (± 0.086) 0.935 (± 0.084) 0.362 (± 0.082) 0.108 (± 0.032) 0.17 (± 0.034) 0.041 (± 0.012) 0.043 (± 0.003) 0.020 (± 0.004) 0.0203 (± 0.0007) 0.003 (± 0.0001)

0.657 (± 0.046) 1.397 (± 0.226) 0.506 (± 0.036) 0.429 (± 0.048) 0.818 (± 0.099) 0.081 (± 0.011) 0.063 (± 0.007) 0.023 (± 0.002) 0.0150 (± 0.0008) 0.0045 (± 0.0003)



575 576

Page 26 of 33

FIGURE LEGEND

577

Figure 1.

Dose/threshold plot showing the concentrations of 230 key food odorants, which by definition exceed their threshold concentration in 227 food samples analyzed,5 plotted against their human threshold concentration. To increase readability, the 230 key food odorants are grouped in different chemical classes.

Figure 2.

Derivatization of carbonyl compounds with 3-nitrophenylhydrazine (3NPH)

using

N-(3-dimethylaminopropyl)-N’-ethylcarbodiimide

hydrochloride (EDC) as catalyst. Figure 3.

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 2methylpropanoate (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),

acetaldehyde-d3 (4-d3), glucose-13C6 (15-13C6), malic acid-d3 (17-d3), and citric acid-13C2 (18-13C2) after 3-NPH derivatization. Signal 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 2methylbutanoate (12), propyl 2-methylbutanaote (13), ethyl hexanoate (14), phlorizin (19), epicatechine (20), chlorogenic acid (21) and the


Page 27 of 33


internal standards β-damascenone-d4 (3-d4), ethyl butyrate-d5 (5-d5), butyl-d5 acetate (6-d5), ethyl 2-methyl-d3 butyrate (7-d3), ethyl propanoate-d5 (9-d5), hexyl-d5 acetate (10-d5), methyl 2-methyl-d3 butyrate (12-d3), ethyl hexanoate-d5 (14-d5), dihydrorobinetin (22), and caffeic acid ethyl ester-d5 (23-d5) after SBSE enrichment (esters) or direct injection (polyphenols). Signal intensity of each mass transition is normalised. 578



579

Hofstetter, Dunkel, Hofmann, Figure 1

580 581

582 583 584 585 586


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Page 29 of 33

587



588 589

590 591 592 593



594


595

596 597 598 599 600 601 602 603 604 605


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606



607 608

609 610 611 612 613 614 615 616 617 618



619


620 621

622


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toc 176x117mm (150 x 150 DPI)


Unified Flavor Quantitation: Towards High-Throughput Analysis of Key

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