standard addition strip for quantitative electrostatic...
TRANSCRIPT
Standard addition strip for quantitative electrostatic
spray ionization mass spectrometry analysis:
Determination of caffeine in drinks.
-Supporting Information-
Elena Tobolkina1, Liang Qiao1 Christophe Roussel2 and Hubert H. Girault1
1: Laboratoire d’Electrochimie Physique et Analytique, Ecole Polytechnique Fédérale de Lausanne (EPFL), Station 6, CH-1015 Lausanne, Switzerland 2 : Section of Chemistry and Chemical Engineering / Institute of Chemical Sciences and Engineering, Ecole Polytechnique Fédérale de Lausanne, Station 6, CH-1015 Lausanne, Switzerland
* CORRESPONDING AUTHOR FOOTNOTE E-mail: [email protected] Telephone number: +41-21-693 3145 Fax number: +41-21-693 3667
SI-1: Limit of detection of caffeine by ESTASI-MS.
5 μl of caffeine (51 nM) in ESI buffer (50% methanol, 49% water and 1% acetic
acid) was deposited on the plastic plate for ESTASI-MS analysis. The mass spectrum
was recorded in a positive ion mode.
Figure SI-1. Mass spectrum of caffeine with the concentration of 51 nM by ESTASI-MS.
100
80
60
40
20
0210205200195190185180
m/z
Rel
ativ
e In
tens
ity (%
)
Caffeine
SI-2: Quantitative analysis from droplets of standard solution by ESTASI-MS
Figure SI-2.1. The mass spectral peak intensity ratio between caffeine and theobromine as a
function of the caffeine concentration obtained by ESTASI-MS. Error bar shows the standard
deviation calculated from three experiments.
[caf
fein
e/th
eobr
omin
e] y = 0.2528x - 0.1403
R! = 0.99855
0
2
4
6
8
10
12
14
0 10 20 30 40 50
Caffeine concentration in solution, !g/ml
SI-3: Standard addition calibration of caffeine in various drinks by ESTASI-MS
or liquid chromatography.
Solutions containing 5-50 μg/mL of caffeine, constant amount of theobromine (final
concentration: 5 μg/ml) and 50 times diluted beverages (Coffee/tea infusions, Coca-
Cola Classic, Coca-Cola Zero, Ice Tea) in the acidic solution (50% methanol, 49%
water and 1% acetic acid) were deposited on the plastic plate for ESTASI-MS
analysis. All mass spectra were obtained in a positive ion mode and the ratios between
caffeine and theobromine peak intensities were used to plot against concentration of
added pure caffeine.
Figure 3.1. Black coffee sample, plot of caffeine/theobromine single protonated peak
intensity ratio as a function of the added caffeine concentrations obtained by ESTASI-MS.
Error bar shows the standard deviation calculated from three experiments
y = 0.0981x + 0.5329 R! = 0.99573
0
1
2
3
4
5
6
-10 0 10 20 30 40 50
Added caffeine concentration, !g/ml
[caf
fein
e/th
eobr
omin
e]
Figure SI-3.2. Coca-Cola Classic sample, the plot of caffeine/theobromine signal intensity
ratio as a function of the added caffeine concentrations obtained by ESTASI-MS. Error bar
shows the standard deviation calculated from three experiments.
Figure SI-3.3. Coca-Cola Zero sample, the plot of caffeine/theobromine signal intensity ratio
as a function of the added caffeine concentrations obtained by ESTASI-MS. Error bar shows
the standard deviation calculated from three experiments.
y = 0.1268x + 0.2287 R! = 0.98692
0
1
2
3
4
5
6
7
8
0 10 20 30 40 50 60
[caf
fein
e/th
eobr
omin
e]
Added caffeine concentration, !g/mL
Coca-Cola Classic
y = 0.1059x + 0.2062 R! = 0.98787
0
1
2
3
4
5
6
7
0 10 20 30 40 50 60
[caf
fein
e/th
eobr
omin
e]
Added caffeine concentration,!g/mL
Coca-Cola Zero
Figure SI-3.4. Ice tea (“Nestea” lemon), the plot of caffeine/theobromine signal intensity
ratio as a function of the added caffeine concentrations obtained by ESTASI-MS. Error bar
shows the standard deviation calculated from three experiments.
Figure SI-3.5. Black tea extract (“Lipton”) sample, the plot of caffeine/theobromine signal
intensity ratio as a function of the added caffeine concentrations obtained by ESTASI-MS.
Error bar shows the standard deviation calculated from three experiments.
y = 0.1379x + 0.1871 R! = 0.98748
0
1
2
3
4
5
6
7
8
0 10 20 30 40 50 60
[caf
fein
e/th
eobr
omin
e]
Added caffeine concentration, !g/mL
Ice Tea
y = 0.1223x + 0.6315 R! = 0.98618
0
1
2
3
4
5
6
7
8
9
0 10 20 30 40 50 60
[caf
fein
e/th
eobr
omin
e]
Added caffeine concentration,!g/mL
Black Tea
Seven samples for black tea as well as for black coffee were prepared with different
amounts of added pure caffeine. Soluble coffee “Nescafe Gold” (2 g) was diluted in
100 ml of boiled water. Black tea leaves “Lipton” (2 g) were poured for 5 minutes
with 100 ml of boiling water and filtered. The sample of black coffee and tea were
diluted 50 times, and pure caffeine was added to the samples to form a final
concentration from 5 to 50 µg/ml. 20 µl of each solution was injected to HPLC, and
separated with a mobile phase of methanol/water 50/50 (v/v) under a flow rate of 0.8
ml/min. The experiments for each sample were repeated 3 times.
Figure SI-3.6. Black tea extract (“Lipton”) sample, the plot of average area of a caffeine peak
as a function of the added caffeine concentrations obtained by HPLC. Error bar shows the
standard deviation calculated from three experiments.
y = 19137x + 93853 R! = 0.99874
0
400000
800000
1200000
0 10 20 30 40 50 60 Added caffeine concentration, !g/mL
Are
a [!
V*s
ec]
Figure SI-3.7. Coffee (“Nescafe Gold”) sample, the plot of average area of a caffeine peak as
a function of the added caffeine concentrations obtained by HPLC. Error bar shows the
standard deviation calculated from three experiments.
Added caffeine concentration, !g/mL
Are
a [!
V*s
ec]
y = 25604x + 143796
R! = 0.99525
0
400000
800000
1200000
1600000
0 10 20 30 40 50 60
Figure SI-3.8.1 Chromatogram obtained for the determination of caffeine in a black tea sample using 50/50 MeOH/water as the mobile phase. The peak at 3.957 min corresponds to caffeine.
EPFL - SBProject Name: DefaultsReported by User: System
Report Method: Detailed Individual Report Printed 09:52:07 01.05.2014
S A M P L E I N F O R M A T I O N
System Acquired By: solution1Unk. Sample Name: 29.04.2014 13:45:19 Sample Type: Unknow n Date Acquired: IPS Acq. Method: 1 Vial: System Processed By: 1 Injection #: 29.04.2014 13:56:20 Date Processed: 5.00 ulInjection Volume: 2487Channel 2 Channel Name: 10.00 MinutesRun Time: 1.00 per secSampling Rate: Channel Desc.:
Sample Set Name: Sample ValuesUsed in Calculations:
AU
0.000
0.002
0.004
0.006
0.008
0.010
0.012
0.014
0.016
0.018
Minutes1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00 10.00
1.63
6
3.16
2
3.95
7
1
2
3
RT(min)
PeakType
Area(!V*sec) % Area Height
(!V) % Height IntegrationType
PointsAcross Peak
StartTime(min)
EndTime(min)
BaselineStart(min)
1.636
3.162
3.957
Unknow n
Unknow n
Unknow n
12022
24168
140819
6.79
13.65
79.55
1129
3759
17493
5.04
16.80
78.16
bb
bb
bb
25
21
37
1.467
3.017
3.750
1.883
3.367
4.383
1.467
3.017
3.750
Figure SI-3.8.2. Chromatogram obtained for the determination of caffeine in a black coffee sample using 50/50 MeOH/water as the mobile phase. The peak at 3.937 min corresponds to caffeine.
EPFL - SBProject Name: DefaultsReported by User: System
Report Method: Detailed Individual Report Printed 09:47:36 01.05.2014
S A M P L E I N F O R M A T I O N
System Acquired By: coffee 1.1 Unk. Sample Name: 30.04.2014 10:16:01 Sample Type: Unknow n Date Acquired: IPS Acq. Method: 1 Vial: System Processed By: 9 Injection #: 30.04.2014 10:36:59 Date Processed: 5.00 ulInjection Volume: 2487Channel 2 Channel Name: 10.00 MinutesRun Time: 1.00 per secSampling Rate: Channel Desc.:
Sample Set Name: Sample ValuesUsed in Calculations:
AU
0.000
0.005
0.010
0.015
0.020
0.025
Minutes1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00 10.00
1.67
1
3.09
4
3.93
7
1
2
3
RT(min)
PeakType
Area(!V*sec) % Area Height
(!V) % Height IntegrationType
PointsAcross Peak
StartTime(min)
EndTime(min)
BaselineStart(min)
1.671
3.094
3.937
Unknow n
Unknow n
Unknow n
140478
32952
177730
40.00
9.38
50.61
12132
5902
23767
29.02
14.12
56.86
bb
bb
bb
45
16
30
1.500
3.017
3.733
2.250
3.300
4.233
1.500
3.017
3.733
Figure SI-2.8.3 HPLC chromatogram of 0.5 mg/ml pure caffeine using 50/50 MeOH/water as the mobile phase.
EPFL - SBProject Name: DefaultsReported by User: System
Report Method: Detailed Individual Report Printed 09:58:28 01.05.2014
S A M P L E I N F O R M A T I O N
System Acquired By: caffeine 5050Unk. Sample Name: 29.04.2014 10:37:33 Sample Type: Unknow n Date Acquired: IPS Acq. Method: 1 Vial: System Processed By: 1 Injection #: 29.04.2014 13:59:09 Date Processed: 5.00 ulInjection Volume: 2487Channel 2 Channel Name: 15.00 MinutesRun Time: 1.00 per secSampling Rate: Channel Desc.:
Sample Set Name: Sample ValuesUsed in Calculations:
AU
0.00
0.20
0.40
0.60
0.80
1.00
1.20
1.40
1.60
Minutes2.00 4.00 6.00 8.00 10.00 12.00 14.00
3.92
8
1
RT(min)
PeakType
Area(!V*sec) % Area Height
(!V) % Height IntegrationType
PointsAcross Peak
StartTime(min)
EndTime(min)
3.928 Unknow n 11406046 100.00 1571351 100.00 bb 43 3.717 4.433
1
BaselineStart(min)
BaselineEnd(min)
Slope(!V/sec)
Offset(!V)
3.717 4.433 4.070304e-003 -1.515248e-002
SI-4: Method of standard addition using strip-ESTASI-MS
20 μl of a beverage (Coca-Cola Classic, Coca-Cola Zero, Ice Tea or black coffee/tea
infusion) were mixed with 25 μl of theobromine (initial concentration 200 μg/ml).
Afterwards, the mixture was diluted to 1 ml by the acidic solution (50% methanol,
49% water and 1% acetic acid). The solution (5 μl) was deposited on the wells of a
plastic strip, containing the spots of caffeine dried from 5 μl of solution with the
concentrations in a range of 5 to 50 μg/ml. The peak intensity ratios between
caffeine/theobromine were obtained by ESTASI-MS to plot the calibration curves.
Figure SI-4.1. Coca-Cola Classic sample, the plotting of caffeine/theobromine signal
intensity ratio as a function of the amount of caffeine in the dried spot by the strip-ESTASI-
MS standard addition method. The standard deviation was calculated from thee experiments
and shown as the error bar.
y = 0.0102x + 0.0974 R! = 0.99448
0
0.5
1
1.5
2
2.5
3
0 50 100 150 200 250 300
[caf
fein
e/th
eobr
omin
e]
Amount of caffeine on the plate, ng
Coca-Cola Classic
Figure SI-4.2. Coca-Cola Zero sample, the plotting of caffeine/theobromine signal intensity
ratio as a function of the amount of caffeine in the dried spot by the strip-ESTASI-MS
standard addition method. The standard deviation was calculated from thee experiments and
shown as the error bar.
Figure SI-4.3. Ice Tea (“Nestea” lemon) sample, the plotting of caffeine/theobromine signal
intensity ratio as a function of the amount of caffeine in the dried spot by the strip-ESTASI-
MS standard addition method. The standard deviation was calculated from thee experiments
and shown as the error bar.
y = 0.0191x + 0.1875 R! = 0.99429
0
1
2
3
4
5
6
0 50 100 150 200 250 300
[caf
fein
e/th
eobr
omin
e]
Amount of caffeine on the plate, ng
Coca-Cola Zero
y = 0.0147x + 0.1042 R! = 0.9965
0
0.5
1
1.5
2
2.5
3
3.5
4
4.5
0 50 100 150 200 250 300
[caf
fein
e/th
eobr
omin
e]
Amount of caffeine on the plate, ng
Ice Tea
Figure SI-4.4. Black tea infusion (“Lipton”) sample, the plotting of caffeine/theobromine
signal intensity ratio as a function of the amount of caffeine in the dried spot by the strip-
ESTASI-MS standard addition method. The standard deviation was calculated from thee
experiments and shown as the error bar.
y = 0.0129x + 0.3351 R! = 0.98915
0
0.5
1
1.5
2
2.5
3
3.5
4
4.5
0 50 100 150 200 250 300
[caf
fein
e/th
eobr
omin
e]
Amount of caffeine on the plate, ng
Black tea
Figure SI-4.5. Mass spectra of Nestea Lemon obtained by strip-ESTASI-MS. The caffeine
amount on the strip was 50 ng. The MS data was obtained in a positive ion mode.
100
80
60
40
20
0
Rela
tive
Inte
nsity
(%)
400350300250200150100
127.24145.24
181.12
195.08
127.24 198.08
325.12
289.08 343.12210.12 361.08
To verify that the previously dried pure caffeine can be fully extracted from the wells
of a strip during ESTASI-MS analysis, series of experiments were performed.
Droplets (5 μ l each) of caffeine/theobromine mixture in the acetic solution
containing different concentrations of pure caffeine (5, 10, 20, 30, 40, 50 μg/ml) and
a fixed concentration of theobromine (5 μg/ml) were deposited on a blank strip and a
strip with previously dried caffeine spots. The mass spectral intensity ratios between
caffeine and theobromine were calculated and plotted as a function of the caffeine
concentration as shown on Figure 4.6. Curve A with a gradient of 0.2528 was
obtained from the blank strip; while curve B with a gradient of 0.5003, almost double
of curve A, was obtained from the strip with previously dried spots containing 25, 50,
100, 150, 200, 250 ng of caffeine, respectively. Such a result indicates that almost all
previously dried caffeine on the spots was quickly dissolved into the acidic droplet for
ESTASI-MS analyses.
Figure SI-4.6. The mass spectral peak intensity ratio between caffeine and theobromine as a
function of the caffeine concentration in the droplets on a blank strip (A) and on a strip
containing previously dried caffeine spots (B). Error bar shows the standard deviation
calculated from three experiments.
y = 0.5003x + 0.2187 R! = 0.99647
y = 0.2528x - 0.1403 R! = 0.99855
0
5
10
15
20
25
30
0 10 20 30 40 50 60
A.
B.
[caf
fein
e/th
eobr
omin
e]
Caffeine concentration, !g/ml
SI-5: Quantification of caffeine in saliva by strip-ESTASI MS
Figure SI-5.1. The plotting of caffeine/theobromine signal intensity ratio as a function of the
amount of caffeine in the dried spot by the strip-ESTASI-MS standard addition method. The
standard deviation was calculated from thee experiments and shown as the error bar. The
saliva sample was collected directly after drinking a cup of coffee from the local cafeteria
y = 0.0296x + 2.2058 R! = 0.99266
0
2
4
6
8
10
12
0 50 100 150 200 250 300
[caf
fein
e/th
eobr
omin
e]
Amount of caffeine on the plate, ng
Coffee from cafeteria