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: hubert.girault@epfl.ch 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