LC-MS With Simultaneous Electrospray and Atmospheric Pressure Chemical Ionization

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.

Switched electrospray and chemical ionization

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).

Simultaneous electrospray and APCI

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.

Coping with high flow rates/fast chromatography

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.