Supporting Information
Paper: Artefact Peaks in Capillary Electrophoresis. Implications on the Analysis
of Glutamate in Protein Hydrolysate Samples.
Authors: Camila Dalben Madeira Campos, Patricia Aparecida de Campos Braga, Felix
Guillermo Reyes Reyes, José Alberto Fracassi da Silva
Scheme 1 – Dissociation of glutamate. The reported dissociation constants (pKa)
values are (1) 2.155, (2) 4.376, and (3) 9.960 for aqueous media [54].
Figure SI1 – Titration curve slope near pI vs. relative permittivity of the pure solvent.
Figure SI2 – Effects of solvent concentration on titration curve slope.
Figure SI3 – Values of pKa for glutamic acid vs. relative permittivity of pure a) protic and
b) aprotic solvents.
11
10
pKa Value
9
8
7
6
5
4
3
2
0
5
10
15
20
25
30
% Acetonitrile (v/v)
Figure SI4 – Influence of acetonitrile concentration on the pKa values for glutamic acid.
Developed methodology for UHPLC-MS/MS
a.) Sample preparation
Due to matrix effects, the quantification was carried by standard addition method.
Six replicates were done sequentially, according to the requirements of the Brazilian
Ministry of Agriculture, Livestock and Food Supply (MAPA).
Initially, a 100-fold dilution was done with the soy sauce samples purchased in the
local market in 1% formic acid aqueous solution. For each replicate, eight standard
solutions ranging from 0 to approximately 5.2 µmol L-1 monosodium glutamate were
prepared. The diluted samples and the standard solutions were mixed 1:1, filtered and
injected in the UHPLC-MS equipment (Agilent, São Paulo, Brazil).
b.) UHPLC-MS settings
LC-MS/MS analysis for quantitation of monosodium glutamate were carried out
in an Agilent 6460 triple quadrupole tandem mass spectrometer coupled with a UHPLC
1290 system with electrospray ionization (ESI) source in the positive ion mode. The
operation conditions were as follows: gas temperature, 250 ºC; gas flow, 10 L min-1;
nebulizer, 25 psi; sheath gas flow, 10 L min-1; sheath gas temperature, 300 ºC;
Capillary voltage, 3.0 kV and nozzle voltage, 1.0 kV. Sample analyses were performed
in the multiple-reaction monitoring (MRM) scan mode, using a dwell time of 50 ms per
channel, under MassHunter software workstations control. The specific precursor ion
monitored was m/z 148.0, concerning the glutamic acid and it was quantified and
identified by monitoring the transitions m/z 148.0→84.2 and m/z 148.0→130.0,
respectively. The optimized mass spectrometric parameters were established:
fragmentor, 80 V and collision energy 4 V for transitions m/z 148.0→130.0 (qualifier
ion) and 16 V for transitions m/z 148.0→84.2 (quantifier ion). Fragment ions with the
highest intensity were chosen for quantification.
The quantitation of glutamic acid in samples of soy sauce was conducted by
standard addition approach, by spiking standard solution in a range of concentration
into the samples. One microliter of each sample was injected into the Zorbax SB-C8
RRHD column (150 mm x 2.1 mm x 1.8 m) and the analytical method was run using a
linear gradient of 1% formic acid and 0.5% HFBA in purified water (mobile phase A)
and 1% formic acid and 0.5% HFBA in acetonitrile UHPLC grade. The initial mobile
phase composition was 40% mobile phase A, increased to 70% after 1.0 minute and
kept constant for one minute. After that, the initial proportion was re-established for one
minute more in order to equilibrate for the next injection. The employee flow was
0.4 mL min-1 and each run time was 4.0 minutes. The column and autosampler
temperatures were maintained at 30 and 10 ºC, respectively. Analytical instrument
control, data acquisition, and data process were performed using MassHunter software
workstations (Agilent Technologies, USA).
Comparison between methods: Analysis of Variance
The results obtained by Capillary electrophoresis, UHPLC-MS and Enzymatic
detection were compared using analysis of variance (ANOVA) tool, with a confidence
level of 0.05. The results can be seen in the tables below.
Table SI1 – ANOVA for premium sample A.
SUMMARY
Groups
Count
Sum
Average
Variance
UHPLC-MS
5
13.31
2.66
0.04012
CE-C4D
9
22.08171 2.49
0.126447517
Enzymatic
3
7.5
2.5
0
df
MS
ANOVA
Source of Variation SS
F
P-value
F critical
Between Groups
0.141636 2
0.070818 0.845904 0.449972 3.738892
Within Groups
1.17206
0.083719
Total
1.313696 16
14
Table SI2 – ANOVA for premium sample B.
SUMMARY
Groups
Count
Sum
Average
Variance
UHPLC-MS
5
26.68
5.34
0.19603
CE-C4D
6
26.98845 4.75
0.502750823
Enzymatic
3
14.1
4.7
0
df
MS
ANOVA
Source of Variation SS
F
P-value
F critical
Between Groups
1.990354 2
0.995177 3.319396 0.074487 3.982298
Within Groups
3.297874 11
0.299807
Total
5.288229 13
Table SI3 – ANOVA for low-cost sample C.
SUMMARY
Groups
Count
Sum
Average
Variance
UHPLC-MS
6
58.66
9.78
1.700866667
CE-C4D
5
48.69449 9.77
0.256036401
Enzymatic
3
28.8
9.6
0.03
Source of Variation
SS
df
MS
Between Groups
0.063856 2
0.031928 0.036628 0.964152 3.982298
Within Groups
9.588479 11
0.87168
Total
9.652335 13
ANOVA
F
P-value
F critical
Table SI4 – Variance analysis for low-cost sample D
SUMMARY
Groups
Count
Sum
Average
Variance
UHPLC-MS
8
88.4
11.05
1.208485714
CE-C4D
6
64.9565 10.90
0.168029962
Enzymatic
3
35.5
11.8
0.003333333
df
MS
ANOVA
Source of Variation SS
F
P-value
F critical
Between Groups
2.082075 2
1.041037 1.566106 0.243334 3.738892
Within Groups
9.306216 14
0.66473
Total
11.38829 16
Simulations of the separation
We have provided the videos for the simulations obtained at different compositions
of the background electrolyte (BGE). In order to run the videos, the software Simul 5
Complex (free to download at http://web.natur.cuni.cz) developed by the group of the
Dr. Bohuslav Gaš must be installed. Run the software and click on PSQ-Player on the
main menu. A new window for the PSQ-Player will open. Click on the open button to
load the chosen PSQ file. Many simulated parameters are loaded in this process, such
as pH, conductivity, ionic strength, etc. On the Simul main window, uncheck these
boxes and let ‘Component 2’ checked in order to visualise the glutamate peak spatial
evolution. The files were named according to the BGE composition. For example, the
file “Glutamate_10% ACN.psq” refers to the simulation using the pKa data obtained by
titration using 10 % of acetonitrile in the BGE. The concentration of glutamate is
5 mmol L-1, except when specified in the file.
PSQ Files Directory
Original files are too big for uploading.
These files can be downloaded through the following link:
https://www.dropbox.com/l/1Q37JWXus0ccvqQJFe6Gfs