Online supplementary material S1:
Supplement to Section 2: a detailed description of Materials and Methods
2.1 Chemicals
The following reference compounds with the indicated purities and suppliers were obtained:
3-(methylthio-) propanal (methional) ≥ 97 %, 3-methylindole (skatole) ≥ 98 %, 2-acetyl-2thiazoline ≥ 96 %, phenylacetaldehyde ≥ 90 %, 3-hydroxy-4,5-dimethyl-2(5H)-furanone
(sotolone) ≥ 97 %, 5-ethyl-3-hydroxy-4-methyl-2(5H)-furanone (abhexone) ≥ 97 %, 3hydroxy-2-methyl-4H-pyran-4-one (maltol) ≥ 99 %, trimethylpyrazine ≥ 99 %, 3-isopropyl-2methoxypyrazine ≥ 97 %, 2,3-diethyl-5-methylpyrazine ≥ 99 %, 4-ethyl-2-methoxyphenol (4ethylguaiacol) ≥ 98 %, 2-methoxyphenol (guaiacol) ≥ 98 %, 2-methoxy-4-vinylphenol (4vinylguaicol) ≥ 98 %, 4-methoxyphenol ≥ 99 %, 4-methoxybenzaldehyde (p-anisaldehyde)
≥ 98 %, 2-furanmethanethiol (2-furfurylthiol) ≥ 98 %, dimethyl trisulfide ≥ 98 %, 2methylbutanoic acid ≥ 98 %, 3-methylbutanoic acid ≥ 99 %, 2-furanmethanol (furfuryl
alcohol) ≥ 98 %, (E)-1-(2,6,6-trimethyl-1-cyclohexa-1,3-dienyl) but-2-en-1-one ((E)-damascenone) 1.1-1.3 wt. %, and oct-1-en-3-one 50% solution in 1-octen-3-ol were from
Aldrich (Steinheim, Germany). Methylpropanoic acid ≥ 99 % was bought from SAFC
(Steinheim, Germany), 2-furaldehyde (furfural) ≥ 99 % and pyridine ≥ 99.8 % from SigmaAldrich (Steinheim, Germany). 4-Hydroxy-3-methoxybenzaldehyde (vanillin) ≥ 99 % was
obtained from ABCR (Karlsruhe, Germany). Indole ≥ 98.5 %, 4-allyl-2-methoxyphenol
(eugenol) ≥ 99 %, butane-2,3-dione (diacetyl) ≥ 99 %, 4-hydroxy-2,5-dimethyl-3(2H)furanone (furaneol) ≥ 99 %, caffeine (Ph Eur, anhydrous) and butanoic acid ≥ 99.5 % were
purchased from Fluka (Steinheim, Germany). 2-Methyl-3-furanthiol was obtained from
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Symrise (Holzminden, Germany) and 3-isobutyl-2-methoxypyrazine ≥ 99 % from Acros
Organic (Geel, Belgium).
The following stable isotope labelled standards were from aromaLAB AG (Freising,
Germany): [2H3]- maltol, [2H2]- furfuryl alcohol, [2H3]-oct-1-en-3-one, [2H3]-methional,
[13C2]- sotolone, [2H3-4]- (E)--damascenone, [2H6]- dimethyl trisulfide, [2H7]- skatole, and
[2H3]- 4-vinylguaiacol. The isotopic labelled standards [2H5]- 4-ethylguaiacol, [2H3]-guaiacol,
2,2-[2H2]-3-methylbutanoic acid, and [2H7]-indole were purchased from DrEhrenstorfer /
CDN-isotopes (Pointe-Claire, Quebec, Canada). [13C6]- Vanillin and [13C2]- butanoic acid
were from Aldrich (Steinheim, Germany).
Dichloromethane p.a. and acetic acid ≥ 99.95 % were obtained from Th. Geyer GmbH & Co.
KG (Renningen, Germany), and -glucuronidase with <7,500 unit/mL sulfatase activity from
Helix pomatia type HP-2 (aqueous solution > 100,000 units/mL) were from Sigma
(Steinheim, Germany).
2.2 Study design
The study design was in concordance with the requirements of the declaration of Helsinki,
and was conducted after consultation of the local ethical committee.
2.3 Volunteers
Volunteers (8 females and 6 males, age range 22-36, mean age 28) were exhibiting no known
illnesses at the time of examination, and consuming their freely chosen meals without any
specified dietary protocol, with the only exception that they were asked to drink no coffee for
at least two and a half days before sample collection. The volunteers were asked to keep
dietary records for two days before they provided the urine sample. Prior to sample collection
and analysis written consent was obtained from all participants providing urine after a full
explanation of the purpose and nature of the study. The volunteers were allowed to withdraw
from the study at any time.
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2.4 Samples
2.4.1 Urine samples
Random urine samples (European urinalysis guidelines - Summary 2000) were collected in
sterile amber glass bottles. The urine samples were processed and analyzed directly after
donation as described below. Dipstick-testing (multi-property strips-testing) was carried out
on all samples to ensure that no urine with abnormal values was included in the experiments
and therefore accounting for possible contamination and illnesses that volunteers were not
aware of.
Multiple test stripes “Combi-Screen PLUS“ from Analyticon Biotechnologies AG
(Lichtenfels, Germany) were used, providing the possibilities to simultaneously test ascorbic
acid, bilirubin, blood, glucose, ketones, leucocytes, nitrite, pH, protein, specific gravity /
density and urobilinogen.
Each test person gave two samples. Before giving the first urine sample, the test persons were
not allowed to drink coffee for two days before sample collection. On the third day of coffee
abstinence the test persons first gave a urine sample and immediately afterwards drank two
cups of coffee. The test persons drank the two cups of coffee within a maximum time of half
an hour. 1.5 hours after giving the first sample, the test persons gave a second urine sample.
The first urine sample of the volunteers is referred to as “control urine” and the second sample
is referred to as “coffee urine” hereafter.
2.4.2 Coffee samples
Commercially available coffee pads (“Senseo™ kraeftig”) containing a mixture of Arabica
and Robusta coffee were brewed with a Senseo™ coffee pad machine. The coffee was either
subjected to analysis (gas chromatography-olfactometry, gas chromatography-mass
spectrometry and stable isotope dilution assay) as described below (2.6.3, 2.7, 2.8 and 2.9) or
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given to the test persons. Each volunteer consumed two cups of 125 mL coffee, each cup
brewed with one coffee pad. One coffee pad contained 7 g ground coffee.
2.5 Measurement of creatinine
A method based on the reaction of Jaffé was used for creatinine measurement in our
experiments using the creatinine kit from Labor + Technik Eberhard Lehmann GmbH (Berlin,
Germany).
2.6 Solvent Extraction and Solvent Assisted Flavor Evaporation of Urine and Coffee
Volatiles
Solvent assisted flavor evaporation (SAFE) (Engel et al. 1999) was applied for isolation of the
volatiles in urine and in coffee. The urine was either subjected to the SAFE directly for
isolation of the volatile fraction, or immediately subjected to hydrolysis with -glucuronidase
prior to SAFE as described below, and subsequently subjected to volatile isolation via SAFE.
2.6.1 Isolation of the volatile fraction of urine without enzymatic hydrolysis
The labeled internal standards dissolved in dichloromethane were added to 50 mL fresh urine.
The standards were added in similar concentrations as present in urine. (These concentrations
were determined in preliminary experiments.) Then, 25 ml of freshly purified
dichloromethane were added, the mixture was stirred for 30 min, and immediately applied for
mild distillation at 50 °C. After distillation of the mixture additional aliquots of 10 mL of
dichloromethane were administered and distillation was re-performed thrice to achieve
complete transfer of the respective odor compounds. The obtained aqueous distillate phase
was additionally extracted thrice with 50 mL of dichloromethane. Then all combined
dichloromethane phases were dried over anhydrous Na2SO4, and finally concentrated to a
total volume of 100 - 200 μL at 50 °C by means of Vigreux-distillation and micro-distillation
(Bemelmans 1979).
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2.6.2 Enzymatic hydrolysis (-glucuronidase assays) of urine
10 mL of an acetic acid – sodium acetate-buffer adjusted to pH5 were added to 10 mL fresh
urine. Then 0.2 mL of the -glucuronidase-solution was added. The mixture was stirred for
15 h at 37 °C. Afterwards, 10 mL of purified dichloromethane and the labelled internal
standards dissolved in dichloromethane were added and the mixture was stirred for 30 min.
Subsequently, the glucuronidase-treated samples were subjected to SAFE distillation
analogous to the outline described above for the untreated samples. Therefore, additional
aliquots of 5 mL of dichloromethane were administered and distillation was re-performed
thrice. The distillate phase was dried over anhydrous Na2SO4, and finally concentrated to a
total volume of 100 - 200 μL at 50 °C by means of Vigreux-distillation and micro-distillation.
2.6.3 Isolation of the volatile fraction of coffee
5 mL coffee brew prepared according to 2.4.2 were added to 5 mL of freshly purified
dichloromethane. If samples were used for quantitation stable-isotopic labeled standards were
additionally added. The mixture was stirred for 30 min.
Subsequently, the coffee samples were subjected to SAFE distillation analogous to the outline
described above (2.6.1) for the urine samples. Therefore, additional aliquots of 5 mL of
dichloromethane were administered and distillation was re-performed thrice. The distillate
phase was dried over anhydrous Na2SO4, and finally concentrated to a total volume of 100 200 μL at 50 °C by means of Vigreux-distillation and micro-distillation.
2.7 High Resolution Gas Chromatography – Olfactometry and Aroma Extract Dilution
Analysis (AEDA) of Coffee Samples
Application of the samples was done by the cold-on-column technique. High resolution gas
chromatography-olfactometry (HRGC-O) was performed with the specifications given in
(Wagenstaller and Buettner 2013). Odorants were screened by sniffing the effluent after gas
chromatographic separation. Odorants were identified by comparison with reference
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substances on the basis of the following criteria: retention indices (RI) on two capillaries of
different polarity (DB-FFAP, DB-5), mass spectra obtained by MS, and odor quality as well
as odor intensity perceived at the sniffing port. Linear retention indices (RIs) of the
compounds were calculated as described in (Van Den Dool and Kratz 1963).
To characterize the most potent odor constituents high resolution gas chromatographyolfactometry was applied. The flavor dilution (FD) factors of the free aroma compounds in
coffee samples were determined by AEDA (Buettner and Schieberle 2001) from the following
dilution series: the original extract of 200 μL was diluted stepwise (1+3, v/v) with
dichloromethane. HRGC-O was then performed on 2 µL of the original extract (FD=1) and of
the respective dilutions using DB-FFAP and DB-5, both in parallels for each sample.
2.8 Two-Dimensional High Resolution Gas Chromatography-Mass Spectrometry (TDHRGC-MS)
A two-dimensional gas chromatographic system (TD-HRGC) was applied with the
specifications given in (Wagenstaller and Buettner 2013). Mass spectra in positive CI mode
(m/z range 35–249) were generated. The intensities of the selected ions of the odorants and
the labelled standards were calculated by MS Data Review, Varian MS-Workstation (Version
6.9; Service Pack 1, Varian, Inc.). Calibration lines of defined mixtures of odorants and
isotopic labeled standards (1:5, 1:3, 1:1, 1:3, 1:5, w/w) were measured and calibration
functions were calculated by using the relative intensities. The concentration of an odorant in
human urine was obtained by calculating the intensities of selected ions for the odorant as
well as for the matching isotopic labeled standard, incorporating the results of the calibration
function and the known amount of isotopic labeled standard added to the sample.
2.9 High Resolution Gas Chromatography-Mass spectrometry (HRGC-MS)
Quantitation of the two acids was done on a one-dimensional HRGC-MS system as the
concentration of the acids was high enough to quantify them without using the two6
dimensional system. Additionally, the one-dimensional HRGC-MS system was used for the
identification of the volatiles with the highest concentrations in the coffee brew. The onedimensional system was a Finnigan Trace GC Ultra (Thermo Electron Corporation / Thermo
Scientific) coupled to a Thermo DSQ Single Quadrupole MS (Thermo Electron Corporation /
Thermo Scientific). The approach was the same as described above (2.7) for the remaining
compounds. The software was Xcalibur Data System (Version 1.4, Thermo Electron
Corporation / Thermo Scientific).
2.10 Sensory analysis of the urine samples
Participants were trained volunteers from the University of Erlangen (Erlangen, Germany)
and Fraunhofer IVV (Freising, Germany), exhibiting no known illness at the time of
examination and with audited olfactory and gustatory function. Seven assessors were
recruited in preceding weekly training sessions and were trained for at least half a year in
orthonasally and retronasally recognizing about 90 selected odorants at different odorant
concentrations according to their odor qualities, and in naming these according to an in-house
developed flavor language. Sensory analyses were performed in a sensory panel room at 21 ±
1 °C. The samples (25 mL) were randomly presented to the sensory panel in covered glass
vessels (capacity 140 mL) for comparative orthonasal evaluation. The participants were asked
to describe the samples. One control urine as well as one coffee urine were assessed by the
seven sensory panel participants. Additionally, one participant also evaluated all the samples
used for quantitation of odorants in urine.
2.11 Statistical analyses
The one-sided paired-sample Wilcoxon signed rank test was used for the comparison of
concentrations of odorants in control urine and urine after the consumption of coffee.
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Buettner, A., & Schieberle, P. (2001). Evaluation of Aroma Differences between HandSqueezed Juices from Valencia Late and Navel Oranges by Quantitation of Key Odorants
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Engel, W., Bahr, W., & Schieberle, P. (1999). Solvent assisted flavour evaporation - a new
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combined chemo-analytical and human-sensory approach: a potential diagnostic strategy.
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