Supplemental Materials
Identification of urinary biomarkers after consumption of sea buckthorn and
strawberry, by untargeted LC-MS metabolomics: a meal study in adult men.
Cătălina S. Cuparencu, Maj-Britt Schmidt Andersen, Gözde Gürdeniz, Simon Stubbe Schou, Maria
Wichmann Mortensen, Arne Astrup, Lars Ove Dragsted
Department of Nutrition, Exercise and Sports, Faculty of Science, University of Copenhagen, Rolighedsvej
30, DK-1958, Frederiksberg C, Denmark
Table S1. Detailed composition of the test meals
Sea Buckthorn
Sucrose (g)
Fructose (g)1
Glucose (g)1
Fiber (g)
Protein (g)
Fat (g)
Water (g)
From
berries
0.5
1.5
2.2
8.4
2.1
4.3
Added
35
120
Strawberry
From
berries
0.2
2.5
2.3
2.3
1.1
0.9
Added
35
12
3.43
120
Control
From
berries
-
Berry content (g)
150
150
-
Total carbohydrates, incl.
fiber (g)
48.4
48.3
43.8
Total amount served (g)
305
309
300
Total energy (kJ)
941
992
938
Energy density (kJ/100g)
309
321
313
Added
35
8.8
0
2.12
4.33
250
1: Glucose and fructose contents are estimated from Tiitinen et al. (j. Agric. Food Chem. 53 2005 1692-1699) and
Dankost (Copenhagen, Denmark)
2: Protein source: whey protein
3: Fat source: canola oil
Table S2. Nutritional composition of the standardized lunch
Content per 100 g
Average content per
ad libitum meal
E%
Energy
1250 kJ
7500 kJ
Protein
10 g
60 g
Carbohydrate
33 g
198 g
Fat
14 g
84 g
-
13.6 %
44.9 %
41.5 %
Table S3. Optimized parameters used for pre-processing the data in mzMine2
Batch step
Raw data import
Mass detection
Chromatogram builder
Negative mode
Chromatogram deconvolution
Isotopic pattern
Join aligner
Duplicate peak filter
Peak list rows filter
Peak finder
Raw data import
Mass detection
Chromatogram builder
Positive mode
Chromatogram deconvolution
Isotopic pattern
Join aligner
Duplicate peak filter
Parameters
Noise level: 15
Min time span (min): 0.01;
Min height: 4.0E1; m/z tolerance: 0.055 mz or 30 ppm
Chromatographic threshold: 95%; Search minimum in RT
range (min): 0.01; Minimum relative height: 10%;
Minimum absolute height: 4.0E1; Min ratio of peak/top
edge: 1.3; Peak duration range (min): 0.01-0.2
m/z tolerance: 0.06 or 30 ppm; Retention time tolerance:
0.01; Monotonic shape; maximum charge: 1
m/z tolerance: 0.06 or 30 ppm; Absolute retention time
tolerance: 0.15; Weight for both m/z tolerance and
retention time tolerance: 10
m/z tolerance: 0.5 or 600 ppm; RT tolerance: 0.15
Min peaks in a row: 5
Minimum peaks in an isotope pattern: 1; m/z range: 501000; RT range: 0-7; peak duration range: 0.01-0.2
Intensity tolerance: 50%; m/z tolerance: 0.06 or 30 ppm;
Absolute retention time tolerance: 0.15
Noise level: 15
Min time span (min): 0.01;
Min height: 4.0E1; m/z tolerance: 0.055 mz or 30 ppm
Chromatographic threshold: 97%; Search minimum in RT
range (min): 0.01; Minimum relative height: 10%;
Minimum absolute height: 6.0E1; Min ratio of peak/top
edge: 1.5; Peak duration range (min): 0.01-0.2
m/z tolerance: 0.06 or 30 ppm; Retention time tolerance:
0.01; Monotonic shape; maximum charge: 1
m/z tolerance: 0.06 or 30 ppm; Absolute retention time
tolerance: 0.15; Weight for both m/z tolerance and
retention time tolerance: 10
m/z tolerance: 0.5 or 600 ppm; RT tolerance: 0.15
Peak list rows filter
Min peaks in a row: 5
Minimum peaks in an isotope pattern: 1; m/z range: 501000; RT range: 0-7; peak duration range: 0.01-0.2
Peak finder
Intensity tolerance: 50%; m/z tolerance: 0.06 or 30 ppm;
Absolute retention time tolerance: 0.17
*m/z: mass to charge ratio; RT: retention time; Intensity: Peak area
Fig. S1. Graphical representation of the PCA scores plot before (left) and after (right) variable selection,
based on variable importance in projection (VIP) scores >1.
* Black – Sea buckthorn; Red – Strawberry; Green – Control.
A. Positive Mode
a. Time interval: 0-1h
Samples/Scores Plot
20
15
Scores on PC 9 (2.28%)
10
5
0
-5
-10
-15
-20
-20
-15
-10
-5
0
5
10
Scores on PC 7 (2.53%)
b. Time interval: 1-2h
c. Time interval: 2-24h
15
20
25
d. Time interval: pooled 24h
Samples/Scores Plot
30
Scores on PC 2 (6.87%)
20
10
0
-10
-20
-30
-30
-25
-20
-15
-10
-5
0
Scores on PC 6 (3.48%)
5
B. Negative Mode
a. Time interval: 0-1h
b. Time interval: 1-2h
c. Time interval: 2-24h
10
15
20
d. Time interval: pooled 24h
Fig. S2. MS/MS of identified compounds
a. Catechin sulphate (level II): m/z 369.02 (-), RT 2.86 of the sample, not matched with standard
b. Furaneol Glucuronide (level I): m/z 305.09 (+), RT 2.24 matched with standard
c. Pelargonidin Glucuronide (level I): m/z 447.098 (+), RT 3.36 matched with standard
d. P-coumaric acid sulphate (level I): m/z 242.997(-), RT 2.90 matched with standard
e.
Dihydrokaempferol Glucuronide (level I): m/z 463.094(-), RT 3.45 matched with standard
f.
Dihydrokaempferol glucuronide (DG), m/z 463.094(-), RT 3.45 vs isomer RT 3.32
g. 1. Furaneol Sulphate (level II): m/z 206.996 (-), RT 1.74 – Sample MSMS
2. Furaneol Sulphate (m/z 206,99(-)) vs Glucuronide (m/z 303.07(-))
h. Mesifurane sulphate (level III): m/z 221.015(-), RT 3.46
i.
Leucopelargonidin Sulphate (level III): m/z 369.033(-), RT 2.68
j.
Cyclohexane carboxylic acid glycine (level I): m/z 186.11(+), RT 3.51 matched with standard
k. 1-cyclohexene carboxylic acid glycine (level I): m/z 184.10(+), RT 3.47 matched with standard
l.
Cyclohexadiene carboxylic acid glycine (level III): m/z 182.08(+), RT 3.40
m. N-Methyl hippuric acid (level I): m/z 194.08(+), RT 3.47 matched with standard
n. Pyrocatechol Sulphate Spiked (level I): m/z 188.985(-), RT 2.10 matched with standard
o. Isorhamnetin glucuronide (level I): m/z 491.089 (-), RT 3.56 matched with standard
p. Dihydroxycyclohexane carboxylic acid (III) : m/z 159.042(-), RT 0.87
q. Protocatechuic acid glucoside (II): m/z 315.075 (-), RT 1.92