Rapid MS/MS analysis with Acoustic Ejection Mass
Spectrometry (AEMS)
Using the SCIEX Echo® MS System to break bottlenecks in quantitative mass spectrometry throughput
Rolf Kern1, Chang Liu2, Chiu Cheong Aw3
1
SCIEX, USA, 2SCIEX, Canada, 3SCIEX, Singapore
The use of a chromatographic separation technique (such as
high pressure liquid chromatography) in line with a mass
spectrometer has been the standard approach for quantitative
LC-MS analysis since the introduction of commercially available
mass spectrometers. Electrospray ionization (ESI) is the mostcommon ionization technique used with LC-MS because of its
broad compound coverage and ionization efficiency whencompared to other approaches like atmospheric pressurechemical ionization (APCI) or matrix assisted laser desorption
ionization (MALDI). Here a newly developed Acoustic Ejection
Mass Spectrometry (AEMS) platform - the SCIEX Echo® MS
System - is described for rapid, chromatography-free MS/MSanalysis.
High-throughput screening (HTS) assays, which are often usedin early stage pharmaceutical research to identify drugcandidates from libraries of many thousands of compounds,
usually rely on data generated using a plate reader. A key
feature of plate readers is that, once the assay is developed,
they can be run quickly, generating a numeric readout for eachwell in an entire plate in less than a minute. But development of
the assay can be very time consuming. Also, the actual output istypically based on a reaction generating an absorption oremission that is caused by the compound of interest, not by thecompound itself, which can lead to specificity issues and cancomplicate troubleshooting. The Echo MS System brings theease of compound tuning, and the specificity of mass
spectrometry, into the throughput realm of plate readers, making
this technology a viable option for HTS research groups.

Key features of the Echo MS System forhigh-throughput quantification• Rapid sample analysis using Acoustic Droplet Ejection andOpen Port Interface• One sample per second for a single analyte, up to threesamples per second in multiplex mode
• Highly reproducible sample injection• Very low carryover• Broad compound coverage using electrospray ionization
featuring the OptiFlow® Turbo V Ion Source for robust andefficient ionization• Industry proven high sensitivity and quantitative robustness
using the SCIEX Triple Quad™ 6500+ LC-MS/MS System• Ease of implementation using SCIEX OS Software• Purpose built for incorporation into HTS workflows; sample
plate tray can be accessed by most robotic arms

Figure 1. High-speed, reproducible quantification. Quantification of
100 nM dextromethorphan injections, across an entire 384-well plate
acquired at 1 sample per second.

• Open software API for incorporation of platform into existing
automated HTS environmentsp1

Technology
Acoustic Droplet Ejection
Acoustic Droplet Ejection platforms have been widely adopted inhigh-throughput screening labs and are used to rapidly,
accurately and precisely transfer nanoliter volumes of liquids
between plates. Acoustic energy is used to create, and thenamplify, a standing wave in liquid in a sample well. As theamplitude of the standing wave increases, a point is reachedwhen a single droplet is ejected from the wave at its central apex
(Figure 2). This is a well characterized process that can be very
tightly controlled, generating consistent volume droplets fromliquid in a plate well on a microsecond time scale. The sampleplate, a standard footprint 384- or 1536-well plate, is articulated
over the fixed position acoustic module by a pair of XY stepper
motors. The stepper motors rapidly move the plate from well towell, allowing each well to be sampled in turn. In the Echo MS
System, the acoustic ejection module is used to very
reproducibly generate sample droplets of approximately 2.5 nL,
depending on the viscosity of the liquid, in an individual ejection
pulse. Additionally, multiple ejection cycles can be sequentiallyrun very quickly, from 200 to 500Hz depending on the sample
liquid, so multiple droplets effectively merge together for a larger
effective ejection volume.

Samplein wellFigure 3. Graphical representation of the Open Port Interface (OPI).
Samples are injected into the Open Port Interface (left) then carriedthrough the capillary to the ESI probe (right) at the mass spectrometer.

ejection, enters the liquid flow and is then carried to the ESI
source. One advantage of the small droplet size being
introduced into the liquid stream is that the significant dilution
can help to minimize matrix suppression effects.
On the Echo MS System, the OPI position is fixed above theacoustic module, so that the sample plate moves from well towell between these elements. The sample plate can move very
quickly and is capable of allowing three wells to be sampled in
one second.
Mass spectrometry

Coupling
fluid (water)

RF Power
Figure 2. Graphical representation of the Acoustic Droplet
Ejection device.

Finally, the liquid stream is vaporized and ionized compounds
are specifically quantified using the SCIEX Triple Quad 6500+
System. Fitted with the OptiFlow Source and the speciallydeveloped transfer line from the OPI, this mass spectrometer
provides industry-proven sensitivity and robustness, and is an
ideal detector for the Echo MS System for high-throughput,quantitative work.

Open Port Interface

Software and automation

The second main component is the Open Port Interface (OPI),
developed in collaboration with Oak Ridge National Laboratory,
which provides a mechanism that allows the capture and transferof the acoustically ejected droplet to the electrospray source ofthe mass spectrometer. The OPI is a pair of concentrically
positioned tubes that are both open at one end (Figure 3). The
outer tube has liquid (carrier solvent) delivered to it by a low
pressure liquid pump. The inner tube is connected, via a transferline, directly to the capillary of the ESI source. The nebulizer gas(GS1) provides an aspirating “pull” that draws the solvent
delivered from the outer tube into the inner tube. The vortex that
results when the carrier solvent makes the turn from the outertube to the inner tube is where the droplet, created by acoustic

The Echo MS System can run as a stand-alone analyzer,
operated through SCIEX OS Software. This provides an easy toimplement platform for high-throughput quantification fromindividual plates. However, the system was purpose built to beincorporated into an existing high-throughput environment. Theplate holder is positioned so that it is easily accessible to arobotic arm. The SCIEX OS Software has a well-documented
instrument control API that allows the system to be controlled bythird-party scheduling software. When analysis of a plate iscomplete, the system will generate a .csv file, containing
automatically integrated peak areas, as well as sample names
and well locations. This file can be automatically imported by aLIMS.
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Quantitative data generation
For an analytical system to be truly quantitative, it must be able
to perform to certain accepted standards. It should be capable ofquantifying across a usable linear dynamic range, it must be
accurate and reproducible, and it should not be prone tosignificant carryover effects from sample to sample. In the caseof a high-throughput system, these capabilities must bemaintained at the required rapid timescale. To illustrate theEcho MS System capabilities, a series of experiments were runusing dextromethorphan as a reference compound, diluted in
10% methanol in water (v/v). Methanol was used as a carrier
solvent, and details of the MRM method conditions are listed inTable 1.

1.95 nM

Blank

0.97 nM

Blank

0.49 nM

Table 1. MRM details for dextromethorphan.

Q1

Q3

Dwell Time(msec)

DPEP

CE

CXP

272.1

215.1

100

40

1031

13

Linearity and carryover
Dextromethorphan was serially diluted, 1:1 in 10% methanol inwater, from 1000 nM to 0.488 nM, and then a 50 µL sample
aliquot was transferred to a 384-well plate (Beckman Life
Sciences 384PP 2.0 Microplate). In addition to the working
standards, 50 µL aliquots of pure diluent were also transferred toother wells in the plate. The calibration curve was run from highTable 2. Calibration curve statistics.

Concentration (nM)

Peak Area

Accuracy

1000

229010103.1

500105386

94.9

250

51665

93.0

125

29791

107.3

62.5

15218

109.6

31.25

6360

91.4

15.625

3530

101.3

7.813

1552

88.6

3.906

912

103.5

1.953

384

85.3

0.977

245

106.

0.488

139

115.3

Figure 4. Evaluating linearity and carryover for quantification. (Top)Calibration curve was run from 1000 nM to 0.488 nM, from high to low
concentration. (Bottom) Raw data from the low end of the calibration
curve, with blanks in between, are shown in acquisition order.

to low, with blanks being run in between each standard. This wasdone to assess both linearity and carryover susceptibility.
The system showed good linearity across the tested range(Figure 4, Table 2), although the low point (0.488 nM) might nothave a high enough S/N to be considered a true LLOQ.
Importantly, there was no observable carryover from even thehighest concentration calibrant (data not shown).

Speed of analysis and precision
To illustrate the native precision of the system while acquiring
data at a rate of one sample per second, 20 mL of
dextromethorphan solution was prepared at a concentration of
100 nM in 10% methanol in water, and 50 L aliquots weretransferred to all wells of a 384-well plate. Data for every sample
well in the plate were acquired in just over six minutes (Figure 5,and detail in Figure 1). MRM peaks obtained from each well
were integrated using SCIEX OS Software. Peak areas werehighly reproducible, with 1.98 %CV across all 384 wells.

p3very reproducible manner, the amount of sample transferred tothe OPI and into the mass spectrometer. The linear range ofdroplet grouping can be matrix dependent, with sticky or viscous
matrices (such as untreated plasma) having a narrower linear
droplet range.

Avg Area: 141,368cps
StdDev: 2,799%cv: 1.98

Figure 5. Reproducibility of injection across a 384-well plate.
Injections of 100 nM dextromethorphan were collected at 1 Hz persample across the full plate. Peak area reproducibility was 1.98 %CV
across the full dataset.

Quantitative droplet generation
A useful aspect of the Echo MS System is the ability to specify,
in the acoustic ejection method, the number of droplets ejectedduring analysis. The droplet size is based on a complex interplayof the frequency of the acoustic energy and the viscosity of theliquid in the sample well. It should be considered a fixed value fora given liquid. Because droplets can be created so quickly, on a
microsecond scale, the ejection of multiple droplets in a group asa single sampling event can have the effect of increasing, in aZoom of 10x, 6 droplet reps

Droplet Number vs Peak Area25

y = 3E-05x + 0.0943
R² = 0.9997

Droplet Count

20

15

10

To demonstrate, a series of ejections were performed using
dextromethorphan. The ejections ranged from one to twenty
droplets, generating a “droplet ladder” (Figure 6, top). Table 3lists the average areas with the %CV obtained for the 10
replicates across the droplet ladder. The plotted linearity isshown in Figure 6. All ejections for this experiment were done
from a single sample well. The ability to quantitatively control thesample introduction volume, which is analogous to an injection
volume in standard HPLC work, can be very helpful duringmethod development.

Table 3. Droplet ladder statistics.

Droplet Count

Average Peak Area% CV

131013.2

1.10261849.6

2.41389629.1

2.51

4118421.0

2.69

5146231.9

1.75

6181091.6

2.017207362.9

2.31

8240954.0

1.91

9270286.5

1.8410305316.6

1.4711330220.0

1.8012359994.5

1.60

13394398.1

1.98

14425327.8

1.4415457841.7

1.82

16495142.4

0.76

17514707.9

2.45

18548093.9

2.07

19575179.7

1.95

20608447.0

2.43

500

100000200000300000400000500000600000700000

Average Peak AreaFigure 6. Linearity of injection. Droplet ladder (bottom) showed areproducible, linear response from 1 through 20 droplets. 10 replicate werecollected for each point on the droplet ladder (top).

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Conclusions
The newly developed Acoustic Ejection Mass Spectrometry(AEMS) system —the SCIEX Echo MS System— has thepotential to dramatically change the field of high-throughput
analysis:• Quantitative ESI MS/MS analysis speed is now much closer tothat of a plate reader, while preserving the ease of method
development, ability to monitor multiple analytes
simultaneously, specificity and dynamic range of mass
spectrometry• Excellent reproducibility of ejection (with 1.98 %CV across all
384-well plate) has been demonstrated• The system’s hardware and software were designed forinclusion in a high-throughput environment. The sample plate
holder is positioned in such a way that standard robot arms
can transfer plates to and from it easily. The open API allows
scheduling software to control the system without the need forhuman intervention.
• Data processing and export can also be automated. After data
for the plate are acquired, they are processed using aquantification method. A .csv file is automatically generated,with information including well number, peak area and sample
name, which can be imported by a LIMS.

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