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THE EXTRACTION OF MAXIMUM INFORMATION FROM INDIVIDUAL ION ARRIVALS AND ITS APPLICATION TO
EXTENDING THE DYNAMIC RANGE OF IMS-OATOF-MS DATA.
Authors: Martin Green*1, Darrell Williams1, Garry Scott1, Tony Gilbert1, Martin Palmer1, Nick Tomczyk1, Keith Richardson1, Mark Wrona2
Affiliations: 1. Waters Corporation, Wilmslow UK, 2. Waters Corporation, Milford, MA, USA
PURPOSE: Interrogate properties of individual ion
arrivals in time of flight MS using fast FPGA
processing.
Digitised ion arrival events within single time of flight transients were processed using the FPGA and the presence of
saturated ADC samples recorded and stored with each point in
the summed spectra.
If only a small percentage of ion arrivals contain saturated
samples minimal distortion will be caused in the final data.
bovine res test old det 2550V
100
bovine res test old det 2550V
100
%
250
Combined high dynamic
range data
High transmission data
Low transmission data
INTRODUCTION
The emergence of orthogonal time-of-flight mass spectrometry
has been made possible, in great part, by the rapid
development of high-speed digital electronics. Developments
include; increased speed and dynamic range of analogue to
digital conversion, high speed data transfer protocols, large
rapidly accessible memory and high-performance field
programmable gate arrays (FPGAs). FPGA technology has
allowed sophisticated data processing to be applied to
individual ion arrivals, enhancing signal-to-noise-ratio and
arrival time precision and enabling information about each ion
arrival to be extracted and stored.
In this paper, fast digital processing is used to examine each
ion arrival and record information in the final mass spectrum.
A method of increasing dynamic range utilising this information
is described.
METHODS
All data were acquired using a Waters SYNAPT G2-Si mass
spectrometer (Figure 1) using an ACQUITY UPLC system.
100
Memory
50
0
957
0
Time
0
Arrival Time
Intensity
Meta data
957
m/z
958
958
m/z
Metadata:
data:Stored
Stored
with
Meta
with
each
intensity
/
time
each intensity / time
pointininspectrum
spectrum
point
Figure 2 Simplified schematic of ADC acquisition architecture.
Figure 2 shows a simplified schematic of the Synapt data recording architecture. Signals from individual ion strikes at the
electron multiplier detector were first digitised and then processed in real time within the FPGA before being summed in onboard memory. In addition to arrival time and intensity information for each transient, “metadata” (Table 1), describing
other properties of the transients may be extracted and associated with each data point in the final summed spectra. These
data are available for subsequent post processing.
Area LHS, RHS
Width at base
Quantiles
Width LHS, RHS
Standard Deviation Skew
Centre of mass
Kurtosis
Maximum
Width FWHM
Saturated points
Table 1 Examples of metadata which may be stored.
Table 1 lists some of the meta data which may be extracted
and stored from individual ion arrivals within each time-offlight transient using the processing power of the FPGA.
In the following example the presence of (or number of) ADC
digitisation samples which exceed the vertical dynamic range
of the ADC (saturated samples) were recorded for each detected ion arrival event and this information stored with each
time-point in the summed spectra.
UPLC Conditions: Small molecule Mix: Verapamil, Suphadimethoxine, Leucine Enkephalin, Caffeine, Acetaminophen.
Column:
Acquity BEH C18 1.7μm 2.1x50mm
Mobile phase: A. Water +0.1% formic acid
B. Acetonitrile +0.1% formic acid
Gradient:
0 to 5mins 10% to 98% B
RECORDING % SATURATION
250
200
150
100
UPLC Conditions: Propanolol in Human Plasma
Figure 3 shows a representation of a
signal produced by multiple ion arrivals
within a single ToF transient. In this
case the signal exceeds the vertical
dynamic range of the ADC of 8bits i.e.
256.
50
Saturation of signals within time of
Time flight transient at high ion input flux
can lead to distortion in intensity and
time of flight measurement.
Figure 3 Ion arrival
Extreme distortion results in errors in
event exhibiting ADC
quantification and mass measurement.
intensity saturation.
0
Column:
Cortecs UPLC C18+ 1.6μm 2.1x50mm
Mobile phase: A. Water +0.1% formic acid
B. Acetonitrile +0.1% formic acid
Gradient:
0 to 4mins 2% to 60% B
4 to 7mins 60 to 95% B
Once summation is finished, each point is marked with a saturation flag if the percentage of saturated events exceeds a preset threshold.
Region of linear
intensity response
0
In addition, calculation and storage of the ratio of these values
can be calculated. For example the ratio of the maxima to the
area for a given transient may be stored.
Figure1. Synapt G2 Si
Rather than record a saturation flag every time saturation has
been detected, the percentage of saturation at each location in
the summed spectra is calculated during summation. The number of saturated samples in an individual ion arrival may be recorded and used to indicate the extent of saturation.
% arrivals saturating ADC
RESULTS: Alternating high and low transmission
data combined point by point based on saturation
flags. 5-10x improvement in dynamic range.
FPGA
150
Relative intensity
METHOD: Record saturated samples within individual
ion arrivals. Calculate and store percentage of
saturated arrivals with individual spectral points.
Create extended dynamic range LC-IMS-MS data.
ADC
%
200
Detector
10
20
Arrival rate (ions / push)
Figure 4. intensity vs average
ion arrival rate
100
35% of all ion arrivals exceed
the dynamic range of the ADC
80
60
40
20
0
10
0
10
20
30
40
time
Figure 6 Chromatogram illustrating transmission switching dynamic range enhancement (DRE).
Using the architecture described, each point in the continuum
spectra carries a record of the percentage of saturated events.
Combining the high- and low-transmission data may be performed directly by replacing flagged points in the high transmission data with corresponding points from the lowtransmission data Figure 9. This may be performed in real time
as data are read from the ADC memory allowing high dynamic
range LC-IMS-MS data to be produced.
RESULTS: LC-IMS-MS
20
Arrival rate (ions / push)
Figure 5. % saturated events
vs average ion arrival rate
Figure 4 shows a plot of relative intensity vs average ion
arrival rate for an 8 bit ADC assuming an average height for a
single ion arrival equivalent to 8 LSB and a Poisson ion arrival
distribution.
a: No DRE
b: With DRE
It can be seen that no significant distortion occurs in the
measurement of response below an ion arrival rate 20 ions /
push. Where ‘push’ refers to a time-of-flight transient recorded
from a single orthogonal sampling of the axial ion beam.
Figure 5 shows a plot of the proportion of ion arrivals which
contain at least one ADC sample which exceeds 8 bits vs the
ion arrival rate. At 20 ions per push approximately 35% of all
ion arrivals are saturated to some degree.
A threshold of >35% saturated events was chosen to
determine when to associate a saturation flag with a location in
the summed spectra.
CONTINUUM DYNAMIC RANGE
ENHANCEMENT (DRE)
The transmission switching method (DRE) has been shown to
increase the dynamic range of time of flight data1.
a: No DRE
Verapamil
Error 15.8ppm
b: With DRE
TO DOWNLOAD A COPY OF THIS POSTER, VISIT WWW.WATERS.COM/POSTERS
4000
RESULTS: PROPRANOLOL IN
HUMAN PLASMA
3000
2000
E
Non-DRE IMS-MS data Figure 10a was acquired at a spectral
rate of 5 spe/sec. In DRE mode Figure 10b, 100ms of full
transmission data and 100ms of 5% transmission data were
recorded at each collision energy. Data points in the 100%
transmission data containing a saturation flag were replaced
by corresponding points from the 5% transmission data scaled
accordingly.
Figure 10a.
100,000pg/ml
Propanolol in Human
plasma. Non –DRE
0
0
E
HDMS was performed. In HDMS the post-IMS, CID
fragmentation energy is switched repeatedly between low and
high values while recording full IMS-MS nested data. High peak
capacity precursor ion and product ion data are produced.
Precursors and products may be associated by both retention
time and IMS drift time or collision cross section.
20000
40000
60000
80000
100000
Concentration pg/ml
Figure 11 quantification curve a) with and b) without DRE
DISCUSSION
In the LC-IMS experiments shown, 100 ms of IMS-MS data was
acquired at each transmission value. 200 individual mass
spectra were recorded within each 100ms IMS-MS nested data
set.
Combining low- and high-transmission data occurs in real time
at a rate of 2000 MS spectra per second to produce a
seamless, continuum data set with up to 10x increase in
dynamic range and improved isotope and fragment ion ratios.
This dramatic post-processing rate is made possible by the
ability of the FPGA extract information from each ion arrival
signal and record this data with each data point in the summed
spectrum.
As FPGA processing power continues to increase, the potential
for more complex calculations within individual time-of-flight
transients increases, improving mass-spectral data quality .
CONCLUSION

FPGA technology allows information from
individual ion arrivals to be extracted and
recorded with final spectra.

Recording the proportion of ADC saturation with
each mass spectral point allows high and low
transmission IMS-MS data to be ‘stitched’ in real
time. 10x dynamic range improvements achieved.

Continuing development of FPGAs will facilitate
more complex processing of individual ion
arrivals, leading to higher quality time-of-flight
data.
Error 3.5 ppm
Verapamil
Figure 10b.
100,000pg/ml
Propanolol in Human
plasma. With –DRE
b) With—DRE
1000
To demonstrate applicability to quantification of small
molecules Propanolol was spiked into protein precipitated
human plasma at a concentration of 100pg/mL to 100,000pg/
mL.
Figure 7 LC-IMS-MS, DT vs RT a) no DRE, b) with DRE
Figures 7 a and b show the results of LC-IMS-MS separation of
a mixture of six small molecules at 500pg on-column without
DRE and with DRE. A scan time of 100ms at full transmission
and 100ms at 5% transmission was used. This results in an
output spectral rate of 200ms / spectrum and an increase of
10x in dynamic range.
Figure 8 shows an extracted mass chromatogram Verapamil
m/z 455.2910 a) no DRE 15.8ppm mass measurement error
and b) with DRE 3.5ppm mass measurement error.
Figure 11 shows a plot of chromatographic response vs concentration (pg/ml) for Propanolol in human plasma without
DRE [a] and with DRE [b] from 100pg/mL to 100,000pg/mL.
The absolute sensitivity has been reduced by 2x for the DRE
data due to the reduced duty cycle of this experiment.
However, the linear dynamic range has been increased by between 5—10x from approx. 2 orders of magnitude to approx. 3
orders of magnitude.
a) Without—DRE
This produces wide dynamic range IMS-MS spectra at a
spectral rate of 5 spec per second and with an overall duty
cycle of approximately 50%.
High and low transmission spectra are acquired on alternate
scans. The high and low transmission data are then combined
into a single, wide dynamic range spectrum replacing saturated peaks with corresponding peaks from the low transmission data scaled appropriately Figure 6.
Previously this method was restricted to peak detected or centroided data. For LC-IMS-MS data up to 2000 spectra / second
are produced making this method impractical to implement in
‘real-time’.
Figure 9
Figure 9 shows a threedimentional LC-IMS-MS contour plot of the full transmission data in Figure 7a. Only
the points containing greater
than 35% of saturated ion arrivals are displayed. These
points are replaced by the corresponding points in the 5%
transmission data and scaled
to give the high dynamic
range data shown in Figure
7b.
Response
ACQUISITION ARCHITECTURE
intensity
OVERVIEW
References
Figure 8. extracted (m/z 455.2910),RT,DT plot m/z 500pg
Verapamil a) without DRE, b) with DRE
1.
Proceedings of the 49th ASMS Conference on Mass Spectrometry and Allied Topics,
Chicago, Illinois, May 27-31, 2001
©2014 Waters Corporation
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