Broader Coverage and Automatic Mass Calibration Using
the TripleTOF™ 5600 System with DuoSpray™ Ion Source
Zack Rhoades and Hesham Ghobarah
AB SCIEX, Foster City, CA, USA and Concord, Ontario, Canada

The DuoSpray™ Ion Source
Traditionally, a limiting step in LC/MS analysis has been that only
one ionization technique can be used at a time. Switchingionization modes often means physically changing the source.
The AB SCIEX DuoSpray™ Ion Source combines the two mostcommon ionization techniques – an Electrospray Ionization (ESI)
probe and an APCI (Atmospheric Pressure Chemical Ionization)probe integrated in a single source housing with Turbo V™
Source functionality. This allows flexibility in method
development and sample analysis for a wider range of analytes.
On the TripleTOF™ 5600 system, this also enables online
automatic mass calibration using the optional Calibrant Delivery
System (CDS).

The TripleTOF™ 5600 System
The AB SCIEX TripleTOF 5600 System is the first accuratemass, high-resolution system to deliver rapid and sensitive
qualitative and quantitative analysis in a single platform. It
provides high sensitivity for low level quantitation with a dynamic
range comparable to a high performance triple quadrupole mass
spectrometer. Using the highly innovative EasyMass™
calibration system, mass accuracy of ~2 ppm RMS can beroutinely achieved with a simple user friendly external calibration.

The TripleTOF 5600 System enables multiple advanced
workflows such as fast acquisition of high resolution product ion
spectra on many precursors in the same scan cycle. Information
dependent acquisition (IDA) can be performed for simultaneousquantitation and qualitative identification. Selectivity in highly
complex samples can be achieved with advanced techniquessuch as real time multiple mass defect filtering and highresolution MRM (MRMHR).
The TripleTOF 5600 system achieves high speed, resolution,sensitivity, mass accuracy, and a wide linear dynamic range allat the same time.

Key Benefits of the DuoSpray™ Source withthe TripleTOF 5600 System• Perform ionization in ESI or APCI without switching sources.
• When combined with CDS, enables fully automatic andunattended EasyMass™ external mass calibration.
• No interference from calibrant ions during data acquisition.
• Flexible configuration enables more efficient method
development and sample analysis, with broader compoundcoverage.
Figure 1. The DuoSpray™ Source. The DuoSpray Source combines
ESI and APCI probes in a single Turbo V™ Source housing for highest
functionality.

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5Flexible Configuration Options Using theDuoSpray™ Ion Source with the TripleTOF™
5600 System

The CDS pump is controlled directly from Analyst® Software andsupports up to 4 different calibration solutions which can be
automatically selected from the software.

EasyMass™ Automatic External Calibration
External calibration is automatically scheduled and run during
sample batches using Analyst® Software. The entire calibration
process is completed in 2 minutes. The software automatically
activates the CDS which introduces the calibration solution
through one of the probes on the DuoSpray™ Source. Data is
automatically acquired, mass calibration is performed, and thesystem calibration automatically updated. The software thenshuts off the CDS and proceeds with the next sample injection.
With this approach, the mobile phase flow is never interrupted.
Furthermore, the calibration solution is never introduced into thesystem during sample data acquisition. This insures thecalibration solution components will not interfere with full scan

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Mass Accuracy (ppm)

The DuoSpray™ Source extends the flexibility for different
workflows and front-end configurations. Sample analysis can be
performed in ESI while the APCI probe is used for calibration, orvice versa. In the first configuration, the column is connected tothe ESI probe inlet while the APCI probe inlet is connected to theCalibrant Delivery System (CDS, Figure 2) for introducing thecalibration solution. This provides ultimate flexibility for assay
conditions, and eliminates the need for valve switching tointroduce the calibrant into the sample stream

4210-1-2-3-4-5020Time (Hours)
406080100Figure 3. Stability of Mass Accuracy for Long Term Operation. 100
hours of acquisition with the TripleTOF™ 5600 System utilizing theCalibrant Delivery System (CDS). Mass Accuracy RMS = 1.69

data acquisition. The operator only needs to specify thefrequency of calibration (e.g. every 20 sample injections).
This method has the added advantage of allowing the system tocalibrate TOF MS and TOF MS/MS modes separately. Thesoftware automatically analyzes the data acquisition method todetermine if MS, MS/MS or both are being used. The correctscan modes are calibrated, without altering ion source conditions
or interrupting mobile phase flow. The result is a user friendly
automated mass calibration with no mechanical valve switchingrequired and no carryover or interference from the calibrant ions.

DuoSpray™ Source Configuration
The ESI and APCI probes enable ionization across a wide rangeof molecular weights and polarity. The combination of bothionization probes in one source housing allows greater flexibility
in method development, sample analysis, and in the case of theTripleTOF™ 5600 system, flexibility in instrument experimental
design.

Figure 2. The Calibrant Delivery System (CDS). The CDS canaccommodate up to four different solutions for calibration and tuning.
Automatic external mass calibration is performed without operator
intervention during sample acquisition.

In Electrospray ionization, the ionized liquid sample is sprayed
into fine droplets. Through the process of desolvation, the liquidfrom the solvent droplets evaporates as the charged ions in thegas phase are pulled into the mass spectrometer. In
Atmospheric Pressure Chemical Ionization (APCI), the sample isintroduced and vaporized using a combination of gas and heat,then ionized using a corona discharge. ESI is applicable to awide range of polar compounds including peptides and heat
labile small molecules, while APCI can be used for less polar
thermally stable analytes.

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The DuoSpray™ Source consists of an ESI probe in the center
based on Turbo V™ Source geometry (Figure 4). The APCIprobe is mounted to the side and also functions as one of theheaters for ESI. The spray voltage (for ESI) and the corona
needle current (for APCI) are always powered. The ionization
mode is simply determined by which probe is used for sample
introduction, resulting in total flexibility based on assay
requirements.

Using CDS in an Acquisition Batch
The Calibrant Delivery System, in conjunction with Analyst®
Software has the ability to insert calibration runs automaticallyinto an acquisition batch. This reduces or eliminates the need toperform additional calibration runs manually as the Auto-Cal
process periodically inserts calibration runs and executes them
unattended. Auto-Cal uses pre-defined reference tables tocalibrate both MS and MS/MS scan modes in between sampleanalysis runs.

Alternative Calibrant Delivery by ESI
In the default CDS configuration, the ESI probe is used forsample analysis and the APCI probe is used to deliver thecalibrant solution. When APCI is required for sample analysis,the ESI probe can alternatively be used for calibrant introduction.

Conclusions• Combining the DuoSpray™ Source with the TripleTOF™
5600 System enables flexible system configuration andEasyMass™ calibration.
• Using the Calibrant Delivery System (CDS), the calibration
solution can be delivered automatically through either ESI orAPCI.
• External mass calibration is performed in two minutes or lessbetween sample injections in a completely automated andunattended manner.
• By using the DuoSpray™ Source, mobile phase flow is neverinterrupted during mass calibration; and the calibrant is neverintroduced during data acquisition. This eliminates any
potential interference from the calibrant ions.

Figure 4. DuoSpray™ Source. The DuoSpray Source is designed with
the ESI probe in the centre and the APCI probe on the left side. One theright side is a heater for rapid desolvation even at higher flow rates, theAPCI probe also functions as the second heater.

For Research Use Only. Not for use in diagnostic procedures.
© 2011 AB SCIEX. The trademarks mentioned herein are the property of AB Sciex Pte. Ltd. or their respective owners. AB SCIEX™ is being used under license.
Publication number: 3470211-01

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