Using FAIMS to Increase Selectivity for LC-MS Analyses

In the development of many LC-MS methods, interferences are commonplace. In an environment in which data must stand up to rigorous scrutiny, such as in the pharmaceutical industry, selectivity of an analytical method is very important. Conditions required for achieving this selectivity, whether they be related to the sample preparation method, the analytical column, or the MS detection, dictate sample throughput.

Method selectivity is advanced by incorporating FAIMS (high-Field Asymmetric waveform Ion Mobility Spectrometry), a technique that is orthogonal to and complementary with both chromatography and mass spectrometry. This on-line gas-phase ion separation delivers reduced chemical background and separation of isobaric endogenous interferences. This paper discusses the new technology, which can be easily implemented into LC-MS work flows to increase laboratory productivity.

What is FAIMS?

Figure 1- Domed FAIMS electrodes (left) and side-toside electrode (right).

Figure 2- Schematic of HPLC-FAIMS-MS system.

Figure 3 -Domed version of FAIMS mounted on a Q-Tof Micro MS (Waters/Micromass) (ion source, gas, and electrical connections not installed).

Figure 4- Domed FAIMS mounted on a Q-Tof Ultima MS (Waters/Micromass) (installation incomplete).

FAIMS is a technology that separates ions at atmospheric pressure. The FAIMS device is mounted between an atmospheric pressure ion source and the entrance orifice to the vacuum chamber of the MS. Two examples of FAIMS electrodes are shown in Figure 1. A typical system incorporating FAIMS into an existing LC-MS system is shown schematically in Figure 2. Figures 3 and 4 illustrate FAIMS mounted on two types of Waters/Micromass (Manchester, U.K.) mass spectrometers.

Three things are important about the technique: First, it separates ions in a manner quite differently from MS; second, it separates ions without loss of sensitivity (ion counts); and third, the ions flow continuously without the gates or drift times associated with conventional ion mobility spectrometry. The simplicity, sensitivity, and selectivity via separation suggest that the technology will be of significant interest to the LC-MS researcher. (A description of the mechanism of separation in FAIMS can be found at www.faims.com.)

FAIMS is compatible with condensed phase separations because it is physically located between the source and the MS, and serves to sample the ion plume for the MS. As shown in Figure 2, an HPLC is connected to an ion source, which may be electrospray ionization (ESI) or atmospheric pressure chemical ionization (APCI). As the compounds are eluted from the LC column they are ionized into a flowing stream of ions. The stream of ions is transported through FAIMS by a carrier gas. Separation of the ions occurs while the ions are carried along between a pair of closely spaced FAIMS electrodes. As shown in the figure, only some selected ions from the mixture are transmitted through FAIMS. This flow of selected ions is transferred to the MS.

Using software to control FAIMS, the analyst selects the ions that are carried through FAIMS by setting two voltages, the dispersion voltage (DV) and the compensation voltage (CV). The dispersion voltage is the peak voltage of a high-frequency asymmetric waveform that is applied across the FAIMS electrodes. The compensation voltage is a dc bias between the electrodes that compensates for the tendency of a given ion to drift toward one of the electrodes. Fortuitously, this CV is compound dependent. By selecting the CV of transmission of the analyte, other ions are removed by collision with the FAIMS electrodes. Background and interfering ions are therefore removed to improve the signal-to-background ratio (S/B) and potentially speed up analysis by reducing the demands on the chromatographic separation.

Quantitative analysis using FAIMS

Figure 5 - Compensation voltage spectrum of a mixture of ions of theophylline and paraxanthine acquired while monitoring m/z 179 (upper trace), and of isotopically labeled theophylline by monitoring m/z 181 (lower trace). Traces are offset for clarity.

A chemical analysis begins with establishing the practical values of DV and CV for the target analyte compound. A standard sample of the analyte is delivered by direct infusion to the ion source. This gives the user an opportunity to optimize operating conditions of FAIMS (and perhaps the MS). During this direct infusion step, the DV is set to the maximum available on the instrument, and the CV is scanned while monitoring the m/z of the target analyte to produce a CV spectrum. The CV at which the maximum signal is obtained is the CV used for subsequent analyses. Unless experimental conditions such as the temperature, type of carrier gas in FAIMS, or gas pressure in FAIMS are changed, the FAIMS will be operated at this newly identified CV for the duration of the analysis. This is not unlike single ion monitoring experiments using the MS.

Theophylline and paraxanthine, metabolites of caffeine, are difficult to quantify because they have the same m/z (and therefore are not distinguished by MS) and are difficult to separate using HPLC. The traces in Figure 5 illustrate the CV spectra taken during direct infusion-ESI of two samples: The upper trace is a mixture of theophylline and paraxanthine, and the lower trace is a sample containing only 15N-labeled theophylline.

After establishing the empirical value of CV for the analyte (e.g., theophylline is transmitted at a CV of 9.8 V, shown in Figure 5) the remainder of the analysis is performed in the usual manner. The HPLC is connected to the ionization source, and samples are injected. Standard samples, including isotopically labeled analogues of the target analyte and unknowns, are processed. Table 1 summarizes experimental conditions for the LC-FAIMS-MS analysis, and Table 2 summarizes quantitative measurements of samples containing 0.25 ng/μL theophylline with different added concentrations of the interfering paraxanthine. The conventional LC-MS results show anomalously high results because paraxanthine coelutes, and has the same mass as theophylline. Despite coelution of the two compounds, the LC-FAIMS-MS results are quantitative.

Development of the procedure for quantitation of theophylline in the presence of paraxanthine using LC-FAIMS-MS required approx. a half-day. HPLC runs were of ~5 min duration without the need to separate theophylline from paraxanthine. Calibration for theophylline was linear over the tested range from 0.1 to 10 ng/μL.

Separation of ions with identical mass-to-charge ratio

Figure 5 illustrates a situation in which FAIMS can be employed to separate species that cannot be distinguished by the MS and may also be difficult to separate by HPLC. A quantitative determination of theophylline is conducted at a CV of 9.8 V, where the ion of theophylline is selectively transmitted, as shown in Figure 5. At this CV, any ions of paraxanthine (having identical m/z to theophylline) originating from the sample will collide with the FAIMS electrodes and not be introduced to the MS. Similarly, paraxanthine is determined at a CV of approx. 13.6 V without interference by theophylline. Since these compounds are not transmitted through FAIMS under identical conditions, development of the LC method is less time-consuming because it is unnecessary to separate these two compounds by LC.

Quantitative determination of perchlorate

This example is selected to illustrate two points. First, detection of ions of low m/z with electrospray ionization is very difficult, if not impossible, because of the intense background ions. Second, perchlorate is a sufficiently important analyte from an environmental point of view that a specialized analytical method (U.S. EPA Method 314, www.epa.gov/safewater/methods/met314.pdf) involving ion chromatography has emerged as the method of choice. It will be shown that the analysis described above for metabolites of caffeine can readily be used for the detection of perchlorate at levels appropriate for environmental monitoring.