Technical
Notes
# 014
Precise Mass Performance of the micrOTOF with either DualSprayer Internal Calibration or Highly Stable External Calibration
The micrOTOF benchtop ESI-TOF system offers excellent mass accuracy over a wide dynamic range. A mass accuracy of 3
ppm is achieved with both internal and external calibration. This note describes the use of a dual sprayer setup on the Apollo
source for the identification of natural products from green tea using exact mass determination.
Introduction
Because of their antioxidative properties flavanoids from plants have gained
increasing interest in the last years.
Different positive health effects are
discussed, such as protection against
cancer and coronary heart diseases
[1], [2]. For tea, flavanoids are of
great importance as they contribute to
the colour and flavour of the tea brew.
The content of flavanols (catechins) in
fresh tea leaves can add up to 30% of
the dry mass. Another major constituent in tea is caffeine with a quantity of
about 4%.
The following application shows the
identification of catechins from green
tea using exact mass determination
with simultaneous consideration of the
true isotopic pattern (TIP).
purpose of internal calibration the
Bruker Apollo source was equipped
with two sprayers, a simple configuration option which is available
without source modification. The
setup is shown in figure 1.
Experimental
The sprayers are mounted opposite to
each other. The position of each nebulizer relative to the mass spectrometer
inlet may be adapted by spacers accor-
The micrOTOF was coupled to an
Agilent 1100 HPLC system. For the
Plant extracts are normally very complex. The greatest inconvenience when
analyzing flavanoids is the lack of
commercially available standards that
complicates the compound identification. Here we show HPLC separation
in combination with precise measurement of both mass and isotopic pattern as useful tools in the identification
of natural compounds.
The micrOTOF offers the possibility
of empirical formula determination
using both accurate mass and statistical isotopic pattern analysis. A mass
precision of 3 ppm can be achieved
with internal calibration. The calibrant
is introduced into the ion source at the
same time as the sample via a second
sprayer. In this way each spectrum can
be internally recalibrated using automated software routines.
Fig. 1: Dual Sprayer setup on the Bruker Apollo source. Left side: Sprayer with low flow
spacer for introduction of calibration mix (2 µL/min). Right side: Sprayer with high flow
spacer for connection of the HPLC flow at 200 µl/min.
Enabling Life Science Tools Based On Mass Spectrometry™
Sample: Hot water was added to a bag
of green tea and brewed for 5 min.
After cooling, the tea was diluted 1:10
in H2O/acetonitrile (90/10).
Results
Fig. 2 shows the separation of catechins and caffeine from green tea.
The corresponding structures and
elemental formulas of the compounds
are illustrated in figure 3. Seven major
signals were detected. Two peaks
were obtained for 291 m/z and 307
m/z. They belong to the two different epimers, catechin and epicatechin
respectively gallocatechin and epigallocatechin.
Fig. 2: Separation of caffeine and flavanols from a green tea brew.
ding to the flow rate. A low-flow spacer
is used for the nebulizer that delivers
the calibration mixture at a flow rate of
2 μl/min, whereas the nebulizer for the
HPLC eluent is fixed on a high-flow
spacer. In order to adjust the nebulizer pressure for the two sprayers independently a second nitrogen pressure
valve was used.
For internal calibration a standard
mixture of perfluoroalkylphosphazine
(ES Tuning Mixture, Agilent) was
used. The calibrant was infused via a
syringe pump.
isocratic post time
(21 min)
Flow:
0.2 ml/min
Injection:
5 μl
UV Detection: 280 nm
MS parameters
Mode:
ESI positive
Nebulizer:
HPLC: 1.4 bar,
syringe pump: 0.4
bar
Dry Gas:
4 l/min at 220°C
The first step in data processing is the
extraction of the spectra. For all compounds, spectra were automatically
detected and averaged over the whole
peak width, a function made possible
by the very stable mass accuracy over
dynamic concentration range. Each
average spectrum was internally recalibrated using the calibration masses
m/z 118.0863, 322.0481, 622.0290
and 922.0098 m/z.
Afterwards elemental formulas were
calculated using the “GENERATE
MOLECULAR FORMULA” option in
DataAnalysis. By default, the elements
C, H, O and N are considered.
Other HPLC and MS parameters are
given in the following:
HPLC conditions
Column:
Prontosil 120-3C18 H 3.0 μm,
53 x 2.0 mm i.d.
Eluents:
A = 0.1 % acetic
acid in water
B = acetonitrile
Gradient:
12% B, 4 min
isocratic (4 min)
to 25% B in 3 min
(7 min)
25% B, 8 min
isocratic (15 min)
to 12% B in 1 min
(16 min)
12% B, 5 min
Fig. 3: Structures and formulas of catechins and caffeine.
Moreover, an intensity-independent
accuracy has the advantage that concentration of sample and calibrant do
not need to be adjusted. Low quantities of the calibrant are sufficient and
minimize the risk of signal suppression and interferences in the spectrum.
The relative intensity of the calibration
peaks is ≤ 5 % in our example.
The results show that the dual sprayer
setup on the micrOTOF works
smoothly. For comparative interest,
alternative ways of data processing
were investigated: Spectra of peak 27 were externally recalibrated using
the calibration equation obtained from
peak no. 1. Secondly, another HPLCMS run was performed under identical
conditions. The spectra of the second
analysis were externally recalibrated
Fig. 4: Average spectrum of peak 3: Coelution of caffeine and catechin. Signal intensity
of catechin is about 1% relative to caffeine.
Other Parameters were:
•
mass tolerance 3 ppm
•
electron configuration even
•
apply nitrogen rule.
The results are summarized in table 1.
The elemental formulas of all expected
compounds could be determined
with a mass error of less than 3 ppm.
Unique results were obtained for the
peaks 1-5. Only peak 6 and 7 gave
more than one formula, the top hit is
listed in table 1. It corresponds to the
result with the best SigmaFitTM (lowest
Sigma value), meaning the best match
for measured and theoretical isotope
pattern (for details see [3]).
The excellent accuracy over a wide
dynamic range is demonstrated with
the spectrum of the co-eluting peaks
number 3 and 4, namely caffeine and
catechin. The averaged mass spectrum
of peak 3 is shown in figure 4. Caffeine
is by far the dominant signal. Catechin
has an intensity of about 4000 cts,
which is approximately 1 % relative to
caffeine. Its mass peak is only visible
if the m/z range of interest is greatly
enlarged. The results for caffeine and
catechin calculated with “GENERATE MOLECULAR FORMULA” are
shown in figure 5. For both compounds
a unique formula with very good mass
precision and fit of the isotope pattern
is obtained, showing that co-elution is
easily handled even if there is a wide
difference in terms of signal intensity.
Table 1: Flavanols and caffeine identified by accurate mass data obtained from the
micrOTOF ESI-TOF MS with internal calibration.
Peak
no.
RT
[min]
Measured
m/z
Calc.
Formula
Calc.
[M+H]+
Sigma
value
Error
[ppm]
Substance
1
2.2
307.08114
C15H15O7
307.08123
0.0070
0.298
Gallocatechin
2
3.1
307.08109
C15H15O7
307.08123
0.0032
0.437
Epigallocatechin
3
4.0
195.08725
C8H11N4O2
195.08765
0.0046
2.056
Caffeine
4
4.0
291.08675
C15H15O6
291.08631
0.0152
-1.505
Catechin
5
6.6
291.08592
C15H15O6
291.08631
0.0053
1.359
Epicatechin
6
7.2
459.09321
C22H19O11
459.09219
0.0007
-2.233
Epigallocatechin
gallate
7
11.0
443.09842
C22H19O10
443.09727
0.0057
-2.590
Epicatechin gallate
Table 2: Comparison of mass errors calculated from 3 different experiments. A: First run;
all spectra internally recalibrated. B: First run; all spectra externally recalibrated using the
calibration coefficients from peak no. 1. C: Second run; all spectra externally recalibrated
with the calibration equation from peak 1 of the first run.
Peak
no.
RT
[min]
Calc.
Formula
A
Error
[ppm]
B
Error
[ppm]
C
Error
[ppm]
Substance
1
2.2
C15H15O7
0.298
0.298
1.396
Gallocatechin
2
3.1
C15H15O7
0.437
0.513
1.900
Epigallocatechin
3
4.0
C8H11N4O2
2.056
1.217
1.147
Caffeine
4
4.0
C15H15O6
-1.505
-1.894
0.235
Catechin
5
6.6
C15H15O6
1.359
2.228
0.972
Epicatechin
6
7.2
C22H19O11
-2.233
-2.244
-3.088
Epigallocatechin gallate
7
11.0
C22H19O10
-2.590
-0.405
-0.819
Epicatechin gallate
1.497
1.257
1.365
Average absolute
Error [ppm]
0.878
0.868
0.916
Standard Deviation
[ppm]
For caffeine and catechin, which are
present in the same LC peak, unique
formulas with a mass error around 2
ppm were calculated simultaneously
and confirmed by SigmaFitTM even
though catechin is detected at a 100fold lower intensity.
Although the dual sprayer setup
works simply and smoothly on the
micrOTOF it should be remembered that similar mass accuracy can
be achieved with external calibration,
because of the extreme inherent stability of micrOTOF accuracy over time.
References
[1] M. Wang et al., Cancer Res. 52
(1992) 1162-1170
[2] M.G.L. Hertog et al., Lancet 342
(1993) 1007-1011
[3] Bruker Daltonics Application
Note ET-07 / LCMS-42
Authors:
Andrea Kiehne, Oliver Räther
Fig. 5: Result from “Generate Molecular Formula” for caffeine and catechin. Mass accuracy with internal calibration is better than 3 ppm.
with the value obtained from peak no.
1 of the first run (20-30 min earlier).
The mass errors from the three different experiments are listed in table 2.
The mass error is ≤ 3 ppm for all
three calibration methods. No significant difference is observed between
internal and external calibration, even
for consecutive LC runs. Here, the
long-term stability of the micrOTOF
is demonstrated resulting in excellent
mass accuracy over time.
Bruker Daltonik GmbH
Conclusion
The Bruker Apollo source can be
easily converted from a single to a dual
sprayer source and vice versa.
Because of the wide dynamic range
of the micrOTOF ESI TOF system it
is not necessary to adjust sample and
calibrant concentrations. The calibrant
can be introduced at a low concentration in order to avoid signal suppression and interferences.
© 2005 Bruker Daltonics
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