Enhanced efficiency of electrospray mass spectrometry: improved sensitivity and
detection limit
Electronic Supplementary Material
Anneli Kruve1, Ivo Leito1, Koit Herodes1, Asko Laaniste1, Rünno Lõhmus2
1
University of Tartu
Institute of Chemistry
Ravila 14a, Tartu 50411
ESTONIA
2
University of Tartu
Institute of Physics
Riia 142, Tartu 51014
ESTONIA
S1
Chemicals
Methanol (J.T.Baker, Deventer, The Netherlands) and pH = 2.8 buffer solution were
used for preparation of LC/MS eluent. The buffer solution was made of 1 mM
ammonium acetate (Fluka Chemie AG, Buchs, Germany) and 0.1% formic acid
(Fluka, Germany) dissolved in water purified with Millipore Milli-Q Advantage A10
(Millipore, USA).
For sample preparation magnesium sulfate (Lach-Ner, Neratovice, Czech Republic)
and sodium acetate (Reahim; Leningrad, now St Petersburg; Soviet Union, now
Russia) and acetonitrile (J.T.Baker, Deventer, The Netherlands) were used.
Acetonitrile was acidified with glacial acetic acid (Lach-Ner, Neratovice, Czech
Republic). Also Primary Secondary Amine (PSA) sorbent (Supelco, Bellefonte, USA)
was used in the QuEChERS method.
Pesticide standard substances were obtained from Dr. Ehrenstorfer GmbH (Augsburg,
Germany). Stock solutions of approximately 1000 mg/kg in acetonitrile were
prepared. For carbendazim the stock solution was 300 mg/kg. The working standard
contained 4 pesticides (carbendazim, thiabendazole, imazalil and methiocarb). For
spiking appropriate dilutions were made.
Post-extraction spiked samples used in this work were prepared according to buffered
QuEChERS method described by Kruve et al [12].
Liquid Chromatography
The spiked extracts and standard solutions were analyzed with Agilent Series 1100
LC/MSD Trap XCT (Agilent Technologies, Santa-Clara, USA). The LC instrument
was equipped with a binary pump, autosampler, thermostated column compartment.
The effluent passed through diode array detector before reaching the mass
spectrometer. The mass spectrometer uses quadrupole ion trap mass analyzer. For
instrument control Agilent ChemStation for LC Rev. A. 10.02 and MSD Trap Control
version 5.2 were used.
Chromatographic separation was carried out on 250 mm long Zorbax Eclipse XDBC18 column, with internal diameter 4.6 mm and particle size 5 μm. An Eclipse XDBC18 12.5 mm long precolumn was used with internal diameter 4.6 mm and particle
size 5 μm. The autosampler injection volume was set to 10 μl. Gradient elution with
methanol and buffer solution (pH = 2.8) was used. The linear gradient started at 20%
methanol and was raised to 100% within 15 min then the column was eluted 7 min
with methanol. After that the methanol content was lowered to 20% in 3 min.
Stabilization time of 7 min was used between injections. Eluent flow rate was 0.8
ml/min. The retention times for carbendazim, thiabendazole, imazalil and methiocarb
were 8.3, 9.5, 13.7 and 16.5 min respectively.
Mass spectrometer
Agilent XCT Ion Trap MS was used. All of the analyses were carried out in positive
mode. The mass spectrometer was operated in the selected reaction monitoring mode
(SRM). Full MS2 spectra were recorded. Each fragmentation was observed
independently in a separate time window. Observed MS2 were independent of the
nebulizer used. MS2 spectra were recorded. For quantitation the following transitions
were followed: 192.0 -> 160.0, 202.0 -> 175.0, 297.0 -> 201.0 and 226.0 -> 169.0 for
carbendazim, thiodicarb, imazalil and methiocarb, respectively.
The MS parameters were first optimized separately for each nebulizer design at the
chromatographic flow rate according to the process described by Kruve et al [14]. The
S2
parameters were separately optimized for both designs – with and without the
capillary C. The parameters are listed in Table A.1.
Signal-to-noise ratios were obtained with Data Analysis software version 5.2, which
calculates noise over the whole chromatogram except the peaks.
Nebulizer configuration
Figure A.1 Cross-sectional view of the developed nebulizer (Design 2).
S3
Optimization of the gas parameters
Both the gas flow in capillary A as well as in capillary C were optimized. For this
optimization one pesticide – imazalil – was used at different solvent compositions. It
was assumed that gas flow rate optima are independent of the analyte used. The
optimization plots are presented in the Figures A.2 ja A.3. It was found for the
nebulizer design 2 that at optimal conditions the gas flow in capillary C should be as
high as possible (due to instrumental limitations) – 300 mL/min – and in capillary A
lowest possible allowed by the software – 3.8 L/min.
The gas flow rate was also optimized for the nebulizer with only one gas supply
(nebulizer design 1) – capillary A. The optimal flow rate was found to be – 28.5
L/min – the highest flow rate possible due to the instrumental limitations.
From these optima it can be seen that the gas flow in capillary C is much more
effective in assisting nebulization than the gas flow in capillary A – a significantly
lower (by ca 7 times) flow rate is sufficient. This demonstrates that even though the
dimensions of the capillaries in the nebulizer are not optimized the inner capillary C
does lead to an improved nebulization.
S4
Figure A.2 Optimization of the gas flow rate in capillary A for a) design 1 without
capillary C and b) design 2 with capillary C (with gas flow from C fixed to 300
ml/min). Each point (intersection of two lines) represents the average of three
measurements.
2.5E+05
2.0E+05
1.5E+05
MS signal
1.0E+05
5.0E+04
22.8
28.5
100%
80%
60%
19
40%
15.2
Capillary A gas flow (l/min)
20%
11.4
20%
7.6
5%
3.8
28.5
5%
0.0E+00
% MeOH in eluent
2.5E+06
2.0E+06
1.5E+06
MS signal
1.0E+06
5.0E+05
100%
80%
60%
40%
19
22.8
Capillary A gas flow (l/m in)
15.2
11.4
7.6
3.8
0.0E+00
% MeOH in eluent
Figure A.3 The MS signal intensity dependence on the gas flow rate in Capillary C at
different effluent compositions. Each point (intersection of two lines) represents the
average of three measurements.
S5
4.E+06
3.E+06
2.E+06
2.E+06
MS Signal
3.E+06
1.E+06
5.E+05
273
218
164
109
55
20
% MeOH
Gas flow rate (ml/min)
5
0
100
80
60
40
0.E+00
From Figure A.2 and A.3 it can be seen that if capillary C is not used (Figure A.2 a),
independent of the eluent composition, in Capillary A the highest possible nebulizer
gas flow rate must be applied in order to achieve the highest sensitivity. On the other
hand, when Capillary C is applied and used in its optimal conditions (optimization
results presented in Figure A.3) the nebulizer gas flow rate in Capillary A (Figure A.2
b) needs to be the lowest possible. It can be seen that increasing gas flow rate in
capillary A in the case of design 2 leads to decrease of sensitivity. This indicates that
gas flow from capillary C alone provides sufficient nebulization and additional gas
from capillary A blows the droplets away from the ionization chamber too fast and
therefore the signal tends to decrease at higher Capillary A gas flow rates.
MS parameter optimization
The MS parameter optimization was carried out via software. The chromatographic
eluent flow rate 0.8 ml/min and the eluent composition corresponding to elution of the
respective pesticide from the column were used.
The optimal MS parameters are described in Table A.1 for both nebulizer designs.
S6
Table A.1 MS parameters optimized for designs 1 and 2. Optimization was carried out at chromatographic flow rates and effluent composition
corresponded to the elution effluent.
Design 1 (nebulizer gas flow rate 28.5 l/min and no Capillary C)
(V)1
Capillary
Skimmer (V)
Cap Exit (V)
Oct 1DC (V)
Oct 2DC (V)
Trap Drive
Oct RF (Vpp)
Lens 1 (V)
Lens 2 (V)
Frag Ampl
Frag Cut Off
1
Cabendazime Thiabendazole
5000
5000
41.48
41.48
88.52
152.46
10.82
11.39
1.23
1.31
36.41
53
118.03
78.69
0
-8.11
-57.21
-100
1.69
1.16
81.84
101
Imazalil
5000
45.66
157.38
13.69
2.62
71.85
83.61
-4.92
-100
0.58
114.9
Design 2 (capillary C gas flow rate 300 ml/min and nebulizer
gas flow rate 3.8 l/min)
Methiocarb
Cabendazime Thiabendazole
5000
2704.92
2762.29
66.56
43.03
59.67
113.11
76.72
129.84
11.39
13.11
16.2
3.18
0.43
0
53
27.48
36.18
83.61
103.28
118.03
0
-4.84
-4.34
-32.13
-85.25
-97.05
0.5
0.65
1.23
113
84.04
104.97
Imazalil
2704.92
50.16
129.84
14.59
1.12
42.42
152.46
-5.33
-74.92
2.12
125.13
Methiocarb
2991.8
43.03
76.72
8.15
1.07
39.3
78.69
-4.84
-92.62
0.34
98.92
Also called Spray Voltage in other MS programs.
S7
Figure A.4 Calibration graphs with different nebulizer constructions (red dots are with Capillay C and green dots are without Capillay C) for
solvent (first row), spiked onion (second row) and spiked garlic (third row).
S8
Figure A.5 Chromatograms for LoD determination in solvent with design 2 for carbendazim (5.0
μg/kg, S/N 18), thiabendazole (0.2 μg/kg, S/N 8.9), imazalil (0.2 μg/kg, S/N 8.0) and methiocarb (5.0
μg/kg, S/N 7.9).
Intens.
x104
2
ANNELI\14091111: EIC160 +MS2(192.0)
1
0
x104
ANNELI\14091113: EIC175 +MS2(202.0)
1.0
0.5
0.0
ANNELI\14091113: EIC201 +MS2(297.0)
4000
2000
0
3000
ANNELI\14091111: EIC169 +MS2(226.0)
2000
1000
0
8
10
12
14
16
18
Time [min]
S9
Figure A.6 Chromatograms for LoD determination in solvent with design 1 for carbendazim (10.0
μg/kg, S/N 20.5), thiabendazole (1.0 μg/kg, S/N 9.2), imazalil (1.9 μg/kg, S/N 6.9) and methiocarb
(50.0 μg/kg, S/N 75.4).
Intens.
ANNELI\28091135: EIC160 +MS2(192.0)
4000
2000
0
ANNELI\28091126: EIC175 +MS2(202.0)
3000
2000
1000
0
ANNELI\28091126: EIC201 +MS2(297.0)
2000
1000
0
3000
ANNELI\28091145: EIC169 +MS2(226.0)
2000
1000
0
8
10
12
14
16
18
Time [min]
S10
Figure A.7 Chromatograms for LoD determination in apple extract with design 2 for thiabendazole
(0.1 μg/kg, S/N 5.9), imazalil (0.1 μg/kg, S/N 7.9) and methiocarb (10.0 μg/kg, S/N 5.8).
Intens.
x104
0.75
ANNELI\14091123: EIC175 +MS2(202.0)
0.50
0.25
0.00
ANNELI\14091123: EIC201 +MS2(297.0)
6000
4000
2000
0
ANNELI\14091119: EIC169 +MS2(226.0)
4000
3000
2000
1000
0
10
12
14
16
18
Time [min]
S11
Figure A.8 Chromatograms for LoD determination in apple extract with design 1 for thiabendazole
(1.0 μg/kg, S/N 12), imazalil (1.0 μg/k, S/N 16) and methiocarb (50.0 μg/kg, S/N 43).
Intens.
ANNELI\28091142: EIC175 +MS2(202.0)
4000
2000
0
ANNELI\28091142: EIC201 +MS2(297.0)
4000
2000
0
1500
ANNELI\28091138: EIC169 +MS2(226.0)
1000
500
0
10
12
14
16
18
Time [min]
S12
Figure A.9 Chromatograms for LoD determination in garlic extract with design 2 for carbendazim
(0.1 μg/kg, S/N 7.3), thiabendazole (0.2 μg/kg, S/N 6.3), imazalil (25.0 μg/kg, S/N 21.7) and
methiocarb (50.0 μg/kg, S/N 1.8).
Intens.
x104
ANNELI\14091114: EIC160 +MS2(192.0)
0.5
0.0
x104
ANNELI\14091103: EIC175 +MS2(202.0)
1.0
0.5
0.0
x104
1.0
ANNELI\14091108: EIC201 +MS2(297.0)
0.5
0.0
ANNELI\14091101: EIC169 +MS2(226.0)
600
400
200
0
9
10
11
12
13
14
15
16
17
Time [min]
S13
Figure A.10 Chromatograms for LoD determination in garlic extract with design 1 carbendazim (25.0
μg/kg, S/N 7.1), thiabendazole (25.0 μg/kg, S/N 17), imazalil (25.0 μg/kg, S/N 6.9) and methiocarb
(50.0 μg/kg, S/N NA).
Intens.
ANNELI\28091131: EIC160 +MS2(192.0)
1000
500
0
6000
ANNELI\28091131: EIC175 +MS2(202.0)
4000
2000
0
ANNELI\28091131: EIC201 +MS2(297.0)
2000
0
ANNELI\28091133: EIC169 +MS2(226.0)
1000
500
0
8
10
12
14
16
18
Time [min]
S14
Figure A.11 Normalized peak areas for the pesticide in spiked garlic extracts in a sequence of
injection number with a) nebulizer design 2 and b) Agilent commercial nebulizer.
S15