Today, liquid chromatography-mass spectrometry with electrospray ionization
(LC-ESI-MS) is increasingly
being used as the technique of choice
for high-throughput pharmaceutical
analysis. LC-ESI-MS offers a desirable
combination of speed, sensitivity,
selectivity, and reliability; it is estimated
that more than 80% of the
compounds analyzed by HPLC with
electrospray ionization generate ions
in sufficient quantity for MS detection.
Those compounds that do not
respond well to electrospray can usually
be ionized by atmospheric pressure chemical ionization (APCI).
Thus, taken together, ESI and APCI
are capable of generating mass spectra
from most HPLC analytes within the
instrument’s mass range.
Typically, a compound is first analyzed
using ESI. If the response is
poor, then the analysis is repeated
with APCI. Previously, this could be
accomplished only by physically disconnecting
and removing the ESI
source and installing the APCI source
in its place, followed by appropriate
adjustment of the mass spectrometer.
Unfortunately, source exchange and
subsequent reanalysis interrupt and
slow the workflow and, from a cumulative
perspective, significantly
reduce analytical throughput. One
solution to this problem is to maintain
multiple instruments with ESI
and APCI sources. In this way, an APCI-MS system is always available
for the determination of analytes that
are unresponsive to ESI. For many
laboratories, maintaining parallel
lines of LC-MS instruments with
respective ESI and APCI sources
would be prohibitively expensive and
operationally burdensome. Moreover,
additional time and personnel would
still be required to repeat the analyses
using APCI whenever electrospray
ionization was ineffective.
Figure 1 - Schematic representation of two different approaches to integrating ESI and APCI in
a source-switchable configuration: a) Mechanical switching—a valve directs the HPLC stream to
either the ESI or APCI nebulizer. b) Voltage switching—voltage across source elements can be
switched between conditions required for ESI or APCI. While both approaches enhance throughput
compared with methods requiring the physical exchange of sources on a single instrument, source
switching imposes serious performance limitations. In the mechanically switched configuration, the
system may not be compatible with low flow rates and narrowly separated peaks, especially if the relatively
lengthy switching time compromises data acquisition so that low-level peaks go undetected or
the reduction in data quality negatively affects retention time precision. In contrast, the voltage-switched
configuration is especially vulnerable to being overwhelmed by high HPLC flow rates
because the system heating capacity is insufficient for complete vaporization of solvent, an APCI
requirement. Here, too, data can be lost during source switching, but the problem is less severe than
for the mechanically switched case.
Prior attempts to eliminate the reduction
in analytical throughput associated
with the physical exchange of ESI
and APCI sources have focused on the
development of source configurations
that provide for real-time switching
between ESI and APCI. In order to be
successful, an integrated source design
must circumvent the essential incompatibility
of ESI and APCI spray and
voltage conditions. Moreover, it must
do so while maintaining a level of performance
comparable to corresponding
dedicated sources. One approach
to solving this problem employs
mechanical switching of the HPLC
eluent between ESI and APCI nebulizers,
which alternately generate and
direct aerosols into the respective ESI
and APCI regions of the integrated
source. A variant of this approach utilizes
a single nebulizer to form the
aerosol and then switches the voltage
settings in the source elements,thereby oscillating between conditions
suitable for ESI or APCI. In either
case, once generated, the ions are
focused on the entrance of the MS
detector. While these innovations
eliminate the need to physically
exchange ionization sources, they also
diminish performance by unduly limiting
HPLC flow rates and restricting
data acquisition in a way that compromises
data quality (Figure 1).
Figure 2 - Schematic representation of multimode ESI-APCI source. Under pressure of the nebulizing
gas, HPLC eluent is forced through the nebulizer, converting the liquid stream into an aerosol. Upon
exiting the nebulizer, the aerosol enters the ESI zone (detail 1) where it is charged by the charging electrode
and then separated from the uncharged component by the reversing electrode. The aerosol then enters the
thermal container (detail 2) where it is completely vaporized by powerful infrared lamps. ESI ions and
neutral analyte molecules then pass into the APCI zone. Ions previously formed by ESI are deflected
around the corona, while remaining neutral molecules pass through and are ionized (detail 3). ESI and
APCI ions are then merged into a single stream and directed to the capillary entrance of the MS detector.
Figure 3 - Contrasting simultaneous and dedicated ESI and APCI operation of the multimode
source. The examples shown here demonstrate the advantage of simultaneous ESI and APCI acquisition.
While peak intensities are somewhat lower in simultaneous mode (acquisition time is distributed across the
ESI and APCI product), sensitivity is more than adequate for detection and quantification. The possibility
of using the simultaneous mode of the multimode source to increase coverage in the sequencing and identification
of peptides from protein digests without sacrificing analytical throughput is shown in (b).
An integrated multimode source
(product no. G1978A, Agilent Technologies,
Palo Alto, CA) is designed
for both simultaneous and independent
ESI and APCI in either positive
or negative mode. The design resolves
ESI and APCI voltage and spray condition
incompatibilities by sequencing
the disparate ionization processes
in a way that separates ESI-generated
ions from nonresponsive neutral
molecules and directs the latter into
the APCI corona discharge. Postgeneration,
the streams of ESI and APCI ions are combined and directed into
the detector (Figure 2). The spectra
shown in Figure 3 demonstrate that
the multimode source, operating in
simultaneous mode, is able to detect
both ESI- and APCI-responsive analytes
without compromising throughput.
Table 1 provides an overview of
the advantage gained with the multimode
source by illustrating the
increase in detection efficiency across
a broad range of analytes. The spectra
shown in Figure 3 demonstrate that
the multimode source, operating in
simultaneous mode, is able to detect
both ESI- and APCI-responsive analytes
without compromising performance
in either mode.
Figure 4 - Simultaneous ESI and APCI acquisition at high HPLC flow fates. Results shown
here indicate that the multimode source solvent vaporization system is more than adequate for the
task of complete solvent vaporization required for APCI, even with aqueous HPLC flow rates up
to and including a 2-mL/min flow rate. This is not the case for the mechanically and voltage-switched
sources (Figure 1) that may require reconfiguration of the HPLC, e.g., the use of split
flows, to remove excess solvent when flow rates are elevated. Moreover, time required for continual
system reconfiguration sacrifices throughput. The increased system complexity can also make it be
more susceptible to pressure variations caused by clogging that can impair retention time reproducibility
and increase downtime required for system refurbishment.
One trend reflective of the desire to
increase analytical throughput is the
use of higher HPLC flow rates (fast chromatography). When flow rates are
increased substantially, measures must
often be taken to prevent the MS source from being swamped with
mobile phase solvent. The technique
traditionally employed for this purpose
is split flow. This approach creates large
amounts of waste solvent, especially in
laboratories operating multiple instruments.
The multimode MS eliminates
the need for split flows because it is
designed to handle high HPLC flow
rates without requiring additional configuration
adjustments. To achieve
proper APCI performance in the multimode
source, all mobile phases leaving
the ESI zone must be completely
vaporized. In the multimode source,
this is accomplished by means of an
insulated containment chamber
equipped with a pair of large infrared
heaters of sufficient capacity to completely
evaporate up to 2 mL/min of
100% water mobile phase (Figure 2).
Figure 4 demonstrates the ability of the
multimode source to handle a wide
range of HPLC flow rates without
needing system adjustment.