Increasing Throughput with Multi-Segment-Injection-Capillary
Electrospray Ionization-Mass Spectrometry (MSI-CESI-MS)
John C. Hudson
SCIEX, Regina, SK, Canada

Introduction
Any process or technique that provides substantial improvementin throughput has the potential to be a disruptive analyticalevent. Multi-segment-injection-capillary electrospray ionizationmass spectrometry (MSI-CESI-MS) has the potential to increase
throughput by at least an order of magnitude.
Capillary zone electrophoresis (CZE) and CESI-MS make thispossible because of inherent higher peak capacities and better
resolution over conventional instrumental techniques such as
GC-MS and LC-MS.

Figure 1. Figure 1. The OptiMS cartridge from SCIEX provides anultra-low flow ESI interface.

MSI is a multiplexing technique in which multiple samples

high-peak capacity, resolution and sensitivity of CESI-MS

are injected onto the capillary prior to applying the separation

technology (Figure 1).

voltage. Each sample is injected followed by a spacer solution,
which provides a differentiating gap between samples in theinjection process. It is important to design the injection process
and MS detection scheme to allow easy identification of analytes
within complex sample output.
The samples are dissolved in an electrolyte, which provides
peak focusing via transient isotachophoresis (t-ITP), resulting
in increased signal to noise. The selected electrolyte has a highmobility cation, such as NH4+, which facilitates the t-ITP event.1

Background
Injection of multiple samples of pharmaceuticals using capillaryzone electrophoresis was reported some years ago by
Veuthey et al.2
Britz-McKibbin and coworkers reported the use of multiple
sample injections in the analysis of metabolomics analytes3and applied the technique in a number of studies. They further
referred to the methods as MSI-CE-MS (multiple segment
injection capillary electrophoresis mass spectrometry).
We report here on an extension to that work resulting in a

To demonstrate the effectiveness of this technique,methamphetamine and its metabolite, amphetamine, werespiked into volunteer urine samples, and were extracted
and reconstituted for analysis.
The multi-segment-injection (MSI) technique was applied using
CESI-MS, and the protocol was evaluated using an 8 or 6 sample
MSI injection approach. Regression analysis was applied tothe data in a proof-of-principle evaluation to assess the potentialof this application.

Materials and methods
Chemicals: All chemicals were reagent grade and werepurchased online from VWR International.
Drug and metabolite standards: Amphetamine,methamphetamine and their D11 deuterated internal standards,
all at 1 mg/mL, were purchased from Cerilliant Corporation(Round Rock, TX). These standard solutions in methanol
were diluted and spiked into volunteer pooled urine samples.
Standard solutions for mass spectrometry and extractions wereprepared at 1 ng/μL in 5 to 50 mM ammonium formate (pH 2.85).

high-throughput protocol that takes advantage of the low-flow,

p1

Urine calibration standards: Urine samples were prepared atCapillary interface

OptiMS cartridge with 90 cm bare fusedsilica capillary, 150 μm OD, 30 μm ID with
conductive emitter tip

CE instrument

CESI 8000 Plus High Performance
Separation-ESI Module

MS instrument 1

SCIEX Triple TOF® 6600 LC-MS/MS Systemwith Analyst® Software 1.7

MS instrument 2

Waters Xevo with MassLynx Software 4.1

ESI voltage

1.25 kVSample introductionHydrodynamic 5 psi for 10 s

Table 2 outline the CESI-MS parameters and multiple reaction

Capillary conditioning

Initial conditioning with MeOH, water, 0.1 N
NaOH, water and BGE

monitoring (MRM) transitions that were used for confirmationand quantitative processing. Samples were prepared using theBackground electrolyte(BGE)

25 mM ammonium formate (pH 2.85)

extraction procedure in Figure 2.

Separation

25 kV, 277 v/cm, 2.3 μamp

Temperatures

Capillary 25 °C; Samples 10 °C

2000 ng/mL. These spiked urine samples were diluted with blankurine to prepare calibrators from 1 to 2000 ng/mL. The samples
were kept at ~4 °C until the time of analysis. Spiked urine
samples and blanks were prepared by liquid-liquid extraction
after the addition of internal standards at 50 ng/mL.
Instrument conditions and extraction protocol: Table 1 andImportant:

Table 1. CESI 8000 Plus System with OptiMS parameters.

• A separation current above 5 μA might cause permanent
damage to the separation capillary.

Compound Name

Parent Daughter Dwell Cone Collision
(m/z)
(m/z)
(V)
(V)

• Generally, please do not apply >2000V to generate
electrospray as it may result in capillary damage.

Amphetamine MRM1

136.11

91.03

0.05

1515

Amphetamine MRM2

136.11

91.03

0.05

1515

Results

D11-Amphetamine

147.17

98.06

0.05

2017

Spiked urine samples for methamphetamine and amphetamine

Methamphetamine
MRM1

150.06

91.07

0.05

2015

analyzed using a liquid-liquid extraction protocol (Figure 2).

Methamphetamine
MRM2

150.06119.09

0.05

20

10

D11-Methamphetamine 161.21

97.05

0.05

2518

Table 2. CESI 8000 Plus System with OptiMS conditions.
To 1 ml of urine (or serum, plasma oral fluid)
1. Add 50 μl of mixed 011 Internal Standards to 1 ml of urine followed 		by 0.2 ml of cone. NH4OH and vortex.

using D11 deuterated internal standards (IS) were prepared andFirst, calibrators and blank extracts were injected using an 8 or
6 sample MSI technique composed of 2 sequential runs. Bothruns included a blank and a blank containing deuterated internal
standards, which showed no significant carryover. The total
analysis time was approximately 80 min for both runs (~5 minper sample). See Figure 3.
Regression analysis on 10 point calibrations in triplicate over3 orders of magnitude were linear, with R2>0.995 for both

2. Add 5 ml of 1-chlorobutane and shake for 10 min.

amphetamine and methamphetamine over the range of

3. Centrifuge at 0 °C for 10 min. at 3000 rpm.

1 to 2000 ng/mL of urine (Figure 4).

4. Evaporate at 40 °C under N2 for 10 min. to remove any NH40H, thenadd 10 μl of 1% HCI in MeOH. Vortex and continue to evaporate
with N2.

MSI-CESI-MS analysis of samples were reconstituted in

5. Add 200 μl of 5 mM BGE to each tube and vortex.
6. Transfer to a 200 μl Microfuge tube (Beckman Coulter).
7. Pressure inject the sample for 10 seconds at 5 psi.
Figure 2. Liquid-liquid extraction protocol for bio-fluids.

200 μL of 25 mM ammonium acetate (pH 4.4) and injectedusing 2 psi pressure injections for 5 s. This corresponds to1.7 nL injected or ~8.5 fg injected from the 1 ng/mL calibrator.
LOD/LOQ was the low calibrator, 1 ng/mL for both amphetamineand methamphetamine.

p2

References
1. Busnel, J.-M. et al. High Capacity Capillary Electrophoresis-Electrospray Ionization Mass Spectrometry: Coupling aPorous Sheathless Interface with Transient-Isotachophoresis.
Anal. Chem. 2010, 82, 9476–9483.

Figure 3. MSI calibrations for amphetamine/methamphetamine 8 and 6
sample runs.

2. Geiser, L.; Rudaz, S.; Veuthey, J.-L. Decreasing analysis
time in capillary electrophoresis: Validation and comparisonof quantitative performance in several approaches.
Electrophoresis 2005, 26, 2293–2302.
3. Kuehnbaum, N.L.; Kormendi, A.; Britz-McKibbin, P.
Multisegment Injection-Capillary Electrophoresis-MassSpectrometry: A High-Throughput Platform for Metabolomics
with High Data Fidelity. Anal. Chem. 2013, 85, 10664–10669.

Figure 4. Regression analysis for amphetamine and methamphetamine.

Conclusions
MSI-CESI-MS techniques show the potential to increase sample
throughput using CESI-MS by an order of magnitude, therefore
decreasing analysis time to less than 5 min per sample.
Method development is straightforward using common
electrolytes combined with t-ITP. This approach provides therequired sensitivity to detect and quantify small drug molecules
and their metabolites at sub-therapeutic levels, less than 2 ng/mLof bio-fluids such as urine.
This approach will be of particular value for routine quantificationsof multiple components in applied toxicology and metabolomics.

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