Discovery of exposure markers in urine for Brassica-containing meals served
with different protein sources by UPLC-qTOF-MS untargeted metabolomics
Maj-Britt Schmidt Andersena, Helene Christine Reinbachb, Åsmund Rinnanb, Thaer Barria, Charlotte
Mithrila, Lars Ove Dragsteda
a
Department of Nutrition, Exercise and Sports, Faculty of Science, University of Copenhagen; cDepartment
of Food Science, Faculty of Science, University of Copenhagen
Corresponding author:
Maj-Britt Schmidt Andersen
Department of Nutrition, Exercise and Sports,
Faculty of Science, University of Copenhagen
Rolighedsvej 30
DK-1958 Frederiksberg C
Email: mbsa@life.ku.dk
Telephone: +4529176809
Fax: 3533 2483
Supplemental material
Table S1 Meal ingredients excluding protein sources
Barleyotto
96.7
Soup
100.7
Pie
77.7
Rapeseed oil
5.8
2.5
8.8
Onion
15
15
15
Apple vinegar
2.5
2.5
2.5
Apple
40
40
-
Apple juice
50
62.5
-
White cabbage
25
25
-
Brussels sprouts
20
20
-
-
1.5
1
60
-
-
Cream cheese
12.5
-
-
Bouillon
150
-
-
Kale
-
125
-
Carrots
-
62.5
-
Parsley roots
-
62.5
-
Chicken stock
-
125
-
Wheat/rye/oat flour
(2:1:1)
Baking powder
-
-
50
-
-
1
Yogurt naturel
-
-
25
Eggs
-
-
45
Skimmed milk
-
-
75
Broccoli
-
-
63
Pointed cabbage
-
-
38
Chicory salad
-
-
20
Wholemeal bread
Garlic
Pearl Barley
Ingredients (in grams per single serving) used in the test meals excluding the protein sources. Foods highlighted in bold
are food ingredients investigated in single food studies.
Fish
Table S2 Ingredients used as protein sources in the meals
Monkfish cheeks
Fillet of cod
Barleyotto
25
-
Soup
58
Pie
-
Smoked mackerel
-
-
26
25
-
-
-
54
-
Vegetarian
Meat
Pig jaws
Smoked saddle of pork
Smoked haunch of venison
25
Fava beans
20
-
-
Brown beech mushroom
120
-
-
Poached egg
-
75
-
Split peas
-
15
-
Cottage cheese
-
-
40
Chanterelles
-
-
20
Ingredients (in grams per serving) used as protein sources for the meat, fish and vegetarian versions of the test meals.
Foods highlighted in bold are food ingredients investigated in single food studies.
Cooking methods for meals:
Barleyotto
The onion was chopped and sautéed in rapeseed oil before adding pearl barley which was left to sizzle for one minute.
Then apple juice was added and one minute after also the bouillon. After simmering for 20 minutes, cream cheese was
added.
Vegetarian version: Fava beans were added to the pearl barley for the last 6-8 minutes of cooking. The brown beech
mushrooms were fried for 2-3 minutes and mixed with the pearl barley before serving.
Meat version: Pig jaws were fried in butter before adding apple juice and water. They were then left to simmer for 45
minutes and added to the pearl barley before serving.
Fish version: Monkfish cheeks were fried in butter for 2 minutes and added to the pearl barley before serving.
All Barleyotto meals were seasoned with salt and pepper and served with bread and a salad of Brussels sprouts, white
cabbage and apple with a mixture of rapeseed oil, apple vinegar, salt and pepper as dressing.
Pie
Flour and baking powder were mixed into a dough with water and yogurt. The dough was rolled, put in a pan and baked
for 15 minutes at 160°C. Onion and garlic were chopped and fried in rapeseed oil. Then, broccoli was cut and added to
the pan with the onion. After beating eggs and milk, the mixture was poured over the vegetables and the pie was baked
for 25 minutes at 150°C. Chicory salad was cut, mixed with the protein source and added on top of the pie before
serving.
Vegetable version: Cottage cheese was mixed with sliced chanterelles
Meat version: Smoked haunch of venison (no preparation)
Fish version: Smoked mackerel (no preparation)
All Pie meals were served with bread and seasoned with salt and pepper.
Soup
Onion and garlic were chopped and sautéed in rapeseed oil before adding peeled and chopped carrots and parsley roots.
Apple juice and chicken stock were added and the mixture was boiled until the vegetables were tender
Vegetarian version: Split peas were boiled until tender. The egg was added to boiling water and poached for 5-8
minutes.
Meat version: Smoked haunch of venison was served with chopped parsley.
Fish version: Fillet of cod was baked in the oven with rapeseed oil.
All Soup meals were seasoned with salt and pepper and served with bread and a salad of Brussels sprouts, white
cabbage and apple with a mixture of rapeseed oil, apple vinegar, salt and pepper as dressing.
Fig. S1 Outline of study design for Single-food studies (SFS). A schedule of meals and urine sampling for each study
day is given on the timeline to the left. The right part of the figure shows the nine SFS meals.
Table S3 Parameters and batch steps used for the processing of samples from the meal study and the single-food studies
(SFS).
Batch step
Raw data import
Chromatogram builder
Negative mode
Chromatogram deconvolution
Isotopic pattern
Join aligner
Duplicate peak filter
Peak finder
Export to csv
Raw data import
Chromatogram builder
Positive mode
Chromatogram deconvolution
Isotopic pattern
Join aligner
Duplicate peak filter
Peak finder
Parameters
Noise level: 5.0E (15); Min time span: 0.01 (0.01);
Min height: 5.0E1 (4.0E1); m/z tolerance: 0.06 (0.055 or 30 ppm)
Chromatographic threshold: 98% (95%); Search minimum in RT range:
0:01 (0.01); Minimum relative height: 1% (10 %); Minimum absolute
height: 5.0E1 (4.0E1); Min ratio of peak/top edge: 1.2 (1.3); (Peak duration
range (min): 0.01-0.2)
m/z tolerance: 0.06 (0.06 or 30 ppm); Retention time tolerance: 0:01 (0.01);
Monotonic shape; maximum charge: 1 (1)
m/z tolerance: 0.06 (0.06 or 30 ppm); Absolute retention time tolerance:
0:12 (0.15); Weight for both m/z tolerance and retention time tolerance: 10
(10)
m/z tolerance: 0.06 (0.5 or 600 ppm); RT tolerance: 0:12 (0.15)
Intensity tolerance: 20% (50%); m/z tolerance: 0.06 (0.06 or 30 ppm);
Absolute retention time tolerance: 0:12 (0.15)
Noise level: 4.0E1 (15); Min time span: 0.01 (0.01);
Min height: 4.0E1 (4.0E1); m/z tolerance: 0.026 (0.055 mz or 30 ppm)
Chromatographic threshold: 98% (97%); Search minimum in RT range:
0:01 (0.01); Minimum relative height: 1% (10%); Minimum absolute
height: 4.0E1 (6.0E1); Min ratio of peak/top edge: 1.2 (1.5); (Peak duration
range (min): 0.01-0.2)
m/z tolerance: 0.026 (0.06 or 30 ppm); Retention time tolerance: 0:01
(0.01); Monotonic shape; maximum charge: 1 (1)
m/z tolerance: 0.026 (0.06 or 30 ppm); Absolute retention time tolerance:
0:17 (0.15); Weight for both m/z tolerance and retention time tolerance: 10
(10)
m/z tolerance: 0.026 (0.5 or 600 ppm); RT tolerance: 0:17 (0.15)
Intensity tolerance: 50% (50%); m/z tolerance: 0.026 (0.06 or 30 ppm);
Absolute retention time tolerance: 0:17 (0.17)
Export to csv
Samples from the meal study were processed in MZmine2.2 and samples from the single-food studies were processed in
MZmine 2.7. The numbers used for SFS are given in brackets.
Table S4 List of unknown PEMs found in the meal study
Meal(s)
m/z
Barleyotto and
Soup
Pie
Soup
Barleyotto
vegetarian meal
Adduct/fragment/parent ion
261.093
RT
(min)
2.01
205.096
255.048
403.229
345.188
191.143
325.167
301.157
287.150
311.153
297.136
306.103
158.088
179.011
1.94
0.93
4.05
3.83
3.58
4.14
4.20
4.09
4.00
3.97
2.64
0.77
0.49
[M+H]+
Pos mode
[M+Na]+
[M+Na]+
Pos mode
Pos mode
Neg mode
Neg mode
Pos mode
Pos mode
Pos mode
Pos mode
Pos mode
Pos mode
Peak
time
3
Present in other
meals
No
3
3
3
3
4
3
No
No
No
No
Yes
No
3
3
3
3-4
3
3
No
No
Yes
No
Yes
No
Barleyotto fish
meal
The meals with highest levels of the feature are listed in the first column. Peak time is the urine sampling point, where
the highest level of the feature was detected. None of the listed PEMs were confirmed in single food studies.