Understanding the Variability of Compound Quantification from Targeted Profiling Metabolomics of 1D-1H-NMR Spectra in Synthetic
Mixtures and Urine with Additional Insights on Choice of Pulse Sequences and Robotic Sampling
Stanislav Sokolenko, Ryan McKay, Eric JM Blondeel, Michael J Lewis, Ben George, David Chang and Marc G Aucoin*
*Corresponding Author: Marc G Aucoin, Department of Chemical Engineering, University of Waterloo, maucoin@uwaterloo.ca
Supplementary information 2: Compounds quantified in urine
Table. 1 Full list of compounds profiled in the urine sample. Mean and standard deviation values
calculated from profiling 11 fully replicated scans (including sample re-insertion) of a single
NMR tube.
Compound
Urea
Creatinine
Hippurate
Citrate
Trigonelline
Trimethylamine N-oxide
Glycine
Formate
Glycolate
3-Indoxylsulfate
Ethanolamine
Glucose
Pyroglutamate
Dimethylamine
Creatine
cis-Aconitate
1,3-Dimethylurate
Taurine
Malonate
3-Aminoisobutyrate
Glutamine
Succinate
Alanine
Carnitine
Histidine
Betaine
Xanthine
π-Methylhistidine
Mean (mM)
29.136
6.894
4.218
1.535
0.844
0.583
0.435
0.384
0.340
0.329
0.268
0.224
0.197
0.168
0.163
0.157
0.130
0.126
0.122
0.117
0.114
0.096
0.085
0.083
0.081
0.081
0.080
0.080
Relative Standard
Deviation (%)
0.81
0.47
1.38
0.77
0.86
0.46
1.40
1.12
7.40
4.95
6.32
6.88
24.96
2.44
5.99
14.53
1.31
17.66
7.04
3.92
33.01
64.26
3.86
13.24
27.48
1.80
9.54
41.98
Acetate
τ-Methylhistidine
4-Hydroxyphenylacetate
Phenylalanine
Methylamine
Asparagine
Dimethyl sulfone
Sucrose
3-Methylxanthine
trans-Aconitate
Lactate
2-Hydroxyisobutyrate
3-Hydroxyisovalerate
Tyramine
1,6-Anhydro-β-D-glucose
Tyrosine
Ethanol
Lysine
Methanol
Hypoxanthine
N,N-Dimethylglycine
Acetone
1-Methylnicotinamide
N-Acetylaspartate
Trimethylamine
Leucine
Valine
Isobutyrate
0.079
0.078
0.073
0.070
0.062
0.053
0.051
0.050
0.050
0.048
0.041
0.041
0.041
0.038
0.037
0.036
0.036
0.036
0.035
0.035
0.032
0.031
0.028
0.021
0.016
0.015
0.013
0.004
2.15
18.09
3.25
14.98
3.40
72.22
1.65
5.72
5.82
22.98
9.13
2.23
17.83
14.10
14.35
9.59
37.99
47.82
1.82
4.82
36.85
3.32
9.93
51.13
41.61
35.75
7.68
16.16
Table. 2 List of compounds profiled in the urine sample that were excluded from the main
analysis as their presence in the sample was deemed ambiguous without confirmation by other
analytical methods. Mean and standard deviation values calculated from profiling 11 fully
replicated scans (including sample re-insertion) of a single NMR tube.
Compound
O-Phosphoserine
Homoserine
Gluconate
Serine
Glycylproline
Arginine
Mannitol
Guanidoacetate
Carnosine
N-Methylhydantoin
Lactose
Glutamate
Methylsuccinate
Tryptophan
N-Acetylglycine
Acetoacetate
Benzoate
Phenylacetate
Succinylacetone
N-Nitrosodimethylamine
Tiglylglycine
2-Oxoglutarate
Imidazole
Anserine
Salicylurate
Xylose
Fucose
Homocystine
O-Acetylcarnitine
N-Acetylglutamate
Methionine
Thymol
Mean (mM)
0.665
0.344
0.313
0.301
0.263
0.213
0.157
0.142
0.103
0.089
0.089
0.088
0.085
0.059
0.055
0.054
0.052
0.051
0.049
0.049
0.048
0.047
0.047
0.045
0.044
0.040
0.040
0.039
0.034
0.033
0.021
0.019
Relative Standard
Deviation (%)
17.70
29.19
45.63
20.53
24.24
23.92
52.94
26.42
21.95
36.77
62.04
27.69
16.73
46.21
49.91
28.38
35.66
69.55
25.17
58.75
21.88
24.10
59.04
23.27
38.69
28.56
42.54
26.65
36.47
33.94
64.89
33.43
Tartrate
Butanone
Isoleucine
Isopropanol
Fumarate
N-Acetylglutamine
0.018
0.017
0.015
0.013
0.008
0.001
17.19
34.06
23.63
22.46
19.78
331.66
Table. 3 Observed mean and standard deviations of compounds profiled in the urine sample but
whose identity could not be confirmed, normalized to creatinine concentration (umol/mmol
creatinine units), and literature values taken from the Human Metabolome Databse (HMDB)
database, version 2.5. HMDB literature concentrations were limited to urine from normal,
healthy adults (18+) reported in the database in umol/mmol creatinine. Information from the
indicated number of sources was compiled and a single grand range presented as overall
minimum and maximum. Where the observed mean did not deviate more than 25% from the
minimum-maximum range found in literature, the observed standard deviation was expressed as
a percent of the range span to illustrate the proportional differences between observation
variability and normal variation between healthy patients
Observed
Mean
Compounds
O-Phosphoserine
Homoserine
Gluconate
Serine
Glycylproline
Arginine
Mannitol
Guanidoacetate
Carnosine
N-Methylhydantoin
Lactose
Glutamate
Methylsuccinate
Tryptophan
N-Acetylglycine
Acetoacetate
(umol/
mmol)
96.49
49.90
45.41
43.75
38.09
30.94
22.83
20.65
14.92
12.94
12.86
12.73
12.28
8.55
8.02
7.83
Observed Standard
Deviation
(% of
(umol/
HMDB
mmol)
range)
17.11
NA
14.52
NA
20.69
23.87
9.13
21.24
9.27
NA
7.40
NA
12.07
14.37
5.52
4.65
3.30
16.51
4.75
47.54
8.04
16.31
3.54
NA
2.04
20.23
3.94
14.03
4.02
NA
2.20
3.29
HMDB Literature
Min
(umol/
mmol)
0.33
NA
4.30
10.40
0.40
0.19
1.12
5.20
0.01
1.00
0.63
NA
0.70
1.32
NA
0.00
Max
(umol/
mmol)
1.05
NA
91.00
53.40
0.40
22.97
85.10
124.00
19.97
11.00
49.90
NA
10.80
29.40
NA
67.00
Number
of
Sources
3
0
6
22
1
25
6
15
12
2
4
0
7
14
0
10
Benzoate
Phenylacetate
Succinylacetone
N-Nitrosodimethylamine
Tiglylglycine
2-Oxoglutarate
Imidazole
Anserine
Salicylurate
Xylose
Fucose
Homocystine
O-Acetylcarnitine
N-Acetylglutamate
Methionine
Thymol
Tartrate
Butanone
Isoleucine
Isopropanol
Fumarate
N-Acetylglutamine
7.59
7.36
7.14
7.10
7.00
6.79
6.78
6.58
6.43
5.86
5.82
5.71
4.94
4.79
3.01
2.75
2.62
2.40
2.23
1.85
1.16
0.20
2.71
5.15
1.80
4.18
1.52
1.62
4.02
1.53
2.47
1.67
2.47
1.52
1.79
1.64
1.95
0.92
0.44
0.82
0.52
0.42
0.23
0.67
1.84
NA
NA
NA
24.49
1.08
NA
14.94
8.83
1.68
12.60
NA
25.12
NA
51.47
NA
1.00
7.46
3.18
NA
0.79
NA
0.10
0.30
NA
NA
0.10
0.18
9.42
5.92
0.01
2.90
6.38
0.21
0.36
1.00
0.15
NA
1.30
0.00
0.40
NA
0.02
NA
147.00
5.07
NA
NA
6.30
150.00
9.42
16.16
28.00
102.00
26.00
0.21
7.50
1.00
3.95
NA
46.00
11.00
16.78
NA
28.90
NA
5
3
0
0
2
18
1
2
7
11
4
1
6
1
17
0
2
2
20
0
10
0