TABLE 1: Comparison of relevant parameters between standard ESI and the current breadboard implementation of Echo-MS. *This comparison was performed on a single-quad SQD2 Waters mass spectrometer. The rest of the data presented in this poster was performed on a QToF G2-XS.

FIGURE 1: Schematics of droplet production via standard ESI (left) and Echo-MS (right). In standard ESI, droplets are produced when a Taylor cone is formed at the end of a fluid capillary under a strong electric field. Using an acoustic transducer, droplets of similar dimensions may be produced directly from a microtitre plate well. When an electric field is applied during ejection, these droplets become charged.

FIGURE 3: Acoustic transfer of liquid without contacting the fluid.

FIGURE 2: A schematic of the transfer tube assembly, used for directing charged material from the 384-well microtiter plate to the mass spectrometer.

FIGURE 4: Breadboard implementation of the AMI system for direct acoustic ionization.

FIGURE 8: The plot of peak height vs the concentration (µM) of Phenylpyruvic acid, R2 = 0.98.

FIGURE 9: The plot of average peak height vs the concentration (µM) of Phenylpyruvic acid in 100%, 50% and 0% Acetonitrile (100% water).

FIGURE 7: The plot of average (over a column, 16 replicates) peak height vs the concentration (µM) of Phenylpyruvic acid.

FIGURE 6: Effect of dilution on the response to the AMI-MS of Phenylpyruvic acid (mol. wt. 164.16) dissolved in Acetonitrile. The time trace shows the data corresponding to 10 columns in a 384 well plate for major molecular ion peak (m/z 163). The mass spectrum shows the major and the minor peaks observed. Upon plotting the concentration vs peak height we observe linearity with good regression co-efficient, R2 (Figure 8).

TABLE 2: Summary of results of AMI-MS of analytes dissolved in Acetonitrile. The samples dissolved in Acetonitrile were uniformly filled (50 µL) using Hamilton Nimbus in 384-well polypropylene Echo-MS qualified plates.

FIGURE 5: AMI-MS for different analytes dissolved in Acetonitrile. a) The time trace showing the data from a 384-well plate run for the major molecular ion; b) Zoom-in of the time trace to see individual peaks; c) Mass spectrum showing the major and the minor peaks; d) Data reproducibility in 384 ejections. Each peak corresponds to 400 bursts equivalent to 10 nL (approximately) of the sample.

Acoustic Mist Ionization of Organic Fluids for High ThroughputMass SpectrometryDeepshika Angrish 1, Jeremy Kowalczyk 1, Lucien Ghislain 1, Eric Hall 1, Rich Ellison 1, Rhys Jones 2, Emmy Hoyes 2, Ian Sinclair 3, Martin Bachman 3, John Wingfield 3 , Sammy Datwani 1 1 Labcyte Inc., San Jose CA, USA; 2 Waters, Wilmslow, UK; 3 AstraZeneca, Discovery Sciences, Alderley Park, Macclesfield, Cheshire, UK

© 2018 LABCYTE INC. All rights reserved. Labcyte® and Echo® are registered trademarks of Labcyte Inc., (“Labcyte”) in the United States and/or other countries.POS-ACMS-1.0

SLAS 2018

IntroductionWe present the development of direct acoustic mist ionization (AMI) mass spectrometry to deliver label-free, high-throughput screening (HTS) platform1. We use a customized Labcyte Echo® acoustic liquid handler to eject electrically charged droplets at rates of up to 1400 transfer-events-per-second directly from a 384-well assay plate into a custom transfer interface where desolvation and ion generation occur. The resulting ions are directed into a Waters MS instrument. Our platform is 10 to 100 times faster than the conventional Mass Spec approaches and eliminates liquid chromatography (LC) separation step.

AMI-MS of Analytes in Organic Solvent (100% Acetonitrile) — Reproducibility & SensitivityWe present the capability of AMI to eject and ionize analytes dissolved in organic solvent (100% Acetonitrile) to be detected by a mass spectrometer to enable one-second analysis.

The Dilution EffectWe demonstrated the effect of dilution in Acetonitrile on the AMI-MS response. For this we selected Phenylpyruvic acid (PPA). A 50 µM solution of the acid was serially diluted to 25, 10, 5, 2.5, 1, 0.5, 0.25 and 0.1 µM with Acetonitrile. 100% Acetonitrile was used as a blank. Upon ejection into the MS, the response corresponding the major molecular ion peak, 163 [M-H]- was observed (Figure 6).

Ionization in Different Solvents Briefly we studied the ionization of PPA and its dilution effect in 100% Acetonitrile, 50% Acetonitrile/water and 100%water. The data is summarized in Figure 8. PPA ionizes best in 100% Acetonitrile and could be detected down to 0.1 µM. However, in 50% Acetonitrile and 100% water, LOD was 10 µM. Notably, the signal intensity was 1.3 to 2 times higher in water (Figure 9) than in 50% Acetonitrile.

Calibration Curve and LODA 24-point calibration curve spanning seven orders of magnitude (5 µM to 0.25 pM) was obtained for Coumachlor dissolved in Acetonitrile. We demonstrated the effect of concentration and sensitivity. Across replicates, we achieved good reproducibility with 25 pM LoD, and a Coefficient of Variation as low as 14% across a 384-well plate. Although we did not achieve the linearity in the full range (5 µM to 0.25 pM), but linearity could be obtained for 10 point curve spanning 2500 pM to 10 pM with R2 value > 0.99 (Figure 11).

Conclusions• AMI-MS uses direct acoustic ionization to deliver ~10 nL sample at 1-second

processing time.

• AMI-MS demonstrates good reproducibility and signal sensitivity with a range of analytes dissolved in Acetonitrile in both the ionization modes.

• The addition of pure Acetonitrile expands capabilities of AMI-MS for high throughput, label-free, LC-free assays with low sample consumption and minimal solvent disposal.

• Next steps include direct comparison of AMI-MS with ESI-MS.

Ion Generation with AMIOur method for acoustic ion generation is conceptually similar to standard electro-spray ionization (ESI) and may be discussed in analogous terms. In ESI, a fluid is pumped through a capillary and subjected to an electric field, producing a Taylor cone which ejects charged droplets (Fig. 1, left). Our method uses ultrasonic pulses delivered from an acoustic transducer to produce a fluid cone and a spray of droplets (Figure 1, right). When an electric field is applied during ejection, those droplets may be charged and get desolvated as it passes through the heated transfer interface to produce ions. Relevant comparison parameters between ESI and our method are presented in Table 1.

This acoustic version of ESI, which we term AMI, uses a modified Echo 555 acoustic dispensing system to generate a mist of droplets in the femtolitre volume range directly from a standard 384-well microtiter plate. These droplets pass through a charge field and ionized analytes are produced as they move through a transfer tube and are directed into the sample cone of mass spectrometer. AMI can ionize a range of small molecules, peptides, and proteins and can generate either +ve or –ve ions depending on the polarity of the charge field.

Parameter ESI AMI

Fluid Cone Base 10 - 50 µm 70 µm

Sample Flow 1 - 100 µL / min 5 µL / min

Droplet Size < 5 µm 1 - 20 µm

Electric Field 600 kV/m 200 kV/m

Inlet Temperature 350oC 200oC

Sensitivity femtomol sub-nanomol*

Analyte Working Conc. Solvent Ionization

modeAve. Peak

Height CV* Mol. Wt. Major m/z observed Minor m/z

Coumachlor 5 µM ACN negative 5.99 E+04 15 342.77 341 [M-H]-

Warfarin 5 µM ACN negative 7.00 E+04 18 308.33 307 [M-H]- 637

Warfarin Sodium 5 µM ACN negative 1.09 E+05 30 330.31 307 [M-H]- 637

Sodium Phenylpyruvate 50 µM ACN negative 2.38 E+04 21 186.14 349 [2M+Na]- 535, 721, 164

Reserpine 5 µM ACN+0.08

%FApositive 3.53 E+05 21 608.68 609 [M+H]+ 551, 523, 703

*Over 384 well plate run ACN = Acetonitrile; FA = Formic acid

Fluid Source (Capillary ID 50 µm)

Capillary OD100 µm

N2(g) Sheath Gas 600 kV/m

Electric Field

Ultrasonic Fluid Ejection Cone

200 kV/mElectric Field

Electrospray Ionization Capillary Source Acoustic Echo-MS Source

Charged Droplets(1 -20 µm)

Charged Droplets(< 5 µm)

5. Reserpine (m.wt. 608.68, Positive ionization)

a) b) c)

4. Sodium phenylpyruvate (m.wt. 186.14, Negative ionization)

a) b) c)

3. Sodium warfarin (m.wt. 330.31, Negative ionization)

a) b) c)

2. Warfarin (m.wt. 308.33, Negative ionization)

a) b) c)

1. Coumachlor (m.wt. 324.77, Negative ionization)

384 wells in 7.03 min, 1 well in 1.1 sec

a)

b)

c)d)

1 2 3 4 5 6 7 8 9 10AB 50 25 10 5 2.5 1 0.5 0.25 0.1 0C PPA conc. (µM)DEFGHIJKLMNO Plate format (using a 384-well plate)P

FIGURE 10: Log (conc.) vs Log (avg. peak height) plot across the full span of Coumachlor conc. 5 µM to 0.25 pM in Acetonitrile (100%).

FIGURE 11: 10-point Calibration Curve covering 2500 pM to 10 nM concentration of Coumachlor in Acetonitrile (100%).

Capillary OD 100

µm

Fluid Source(Capillary ID 50 µm)

1254-E