Sensitivity “Hot Spots” in the Direct Analysis in Real Time Mass Spectrometry of Nerve Agent Simulants
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Presented here are findings describing the spatial-dependence of sensitivity and ion suppression effects observed with direct analysis in real time (DART). Continuous liquid infusion of dimethyl methyl phosphonate (DMMP) revealed that ion yield “hot spots” did not always correspond with the highest temperature regions within the ionization space. For instance, at lower concentrations (50 and 100 μM), the highest sensitivities were in the middle of the ionization region at 200 °C where there was a shorter ion transport distance, and the heat available to thermally desorb neutrals was moderate. Conversely, at higher DMMP concentrations (500 μM), the highest ion yield was directly in front of the DART source at 200 °C where it was exposed to the highest temperature for thermal desorption. In matching experiments, differential analyte volatility was observed to play a smaller role in relative ion suppression than differences in proton affinity and the relative sampling positions of analytes. At equimolar concentrations sampled at the same position, suppression was as high as 26× between isoquinoline (proton affinity 952 kJ mol–1, boiling point 242 °C) and p-anisidine (proton affinity 900 kJ mol–1, boiling point 243 °C). This effect was exacerbated when sampling positions of the two analytes differed, reaching levels of relative suppression as high as 4543.0× ± 1406.0. To mitigate this level of relative ion suppression, sampling positions and molar ratios of the analytes were modified to create conditions in which ion suppression was negligible.
Key wordsDirect analysis in real time (DART) Ambient MS Ionization fundamentals Ion suppression Nerve agent simulants
The growth of the ambient desorption/ionization mass spectrometry (MS) field has been rapid, with various different ionization techniques and corresponding applications being reported in a short time span [1, 2, 3]. Ambient MS techniques enable: (1) surface ionization in the absence of enclosures, (2) direct ionization with minimum sample pretreatment, (3) interfacing to most types of mass spectrometers fitted with differentially-pumped atmospheric pressure interfaces, and (4) soft ion generation (low internal energy deposition) such that the majority of detected ions are intact. A subset of techniques in the ambient MS field revolves around direct and alternating current (DC and AC) plasma-based approaches involving chemical ionization (CI) mechanisms. Although several ambient plasma-based ionization techniques have been developed since the introduction of direct analysis in real time (DART) , DART remains the most prevalently studied and used ambient plasma ionization technique due to its commercial availability with over 70 published references within the past two years alone.
Despite the wide adoption of DART as a high-throughput screening tool for pharmaceuticals [5, 6, 7], homeland security [8, 9, 10, 11], metabolomics [12, 13], and polymer analysis [14, 15], the fundamental variables that affect DART analysis are still under investigation. The ionization region in DART MS is a dynamic environment involving complex fluid dynamics, steep temperature gradients, and weak electrostatic fields that are a result of the ion source geometry, sample position and orientation, and instrumental settings . In situations where only qualitative information is required, spatial effects can be largely ignored. However, when highly reproducible and/or quantitative experiments are desired, sample positioning becomes critical to ensure reproducibility of the ionization conditions. Ionization may proceed through several atmospheric pressure reaction pathways involving the primary reactive species formed by DART (metastables) and secondary reactive species such as protonated species from atmospheric gases. The factors that affect the prevailing ionization pathway depend on the presence and type of sample matrix, solvents and contaminants, the DART gas composition, and the chemical properties of the analyte . Even with optimized parameters, there is some evidence that DART may sometimes succumb to deleterious matrix effects involving ion suppression  when the depletion point of reactive species has been reached . Ion suppression is expected to be present to some extent in all ion direct generation approaches, and affect sensitivity and dynamic range by preferentially decreasing analyte signals. The major DART conditions to consider when mitigating ion suppression include the rates of neutral desorption (related to the temperature and flow rate of the DART gas and analyte boiling point, BP), ionization efficiency (proton affinity, PA), and molar ratios of the analytes of interest respect to interfering species. These effects are confounded with the sampling position and orientation in space. From our past experience with DART, we believe that the desorption and ionization processes have differential magnitudes relative to the sample position in space resulting in convoluted ion intensities and relative levels of ion suppression [13, 16]. To study what physicochemical processes and DART conditions contribute to sensitivity, ionization efficiency, ion suppression, and dynamic range, a series of steady-state spatially-resolved experiments were devised to study these effects in more detail.
All reagents were analytical grade (Sigma-Aldrich, St. Louis, MO, USA) and used without further purification. Solutions of dimethyl methylphosphonate (DMMP, 97%), p-anisidine (99%), and isoquinoline (97%) were prepared in nanopure water (Barnstead International, Dubuque, IA, USA). Ultra high purity helium (99.999%; Airgas, Atlanta, GA, USA) was used for the DART glow discharge gas.
MS analysis was performed with a commercial DART-100 ionization source (IonSense, Inc., Saugus, MA, USA) coupled to a quadrupole-time of flight (Q-TOF) mass spectrometer (Bruker micrOTOF-Q I; Bremen, Germany). The Q-TOF mass spectrometer interface was modified to accommodate for the gas flow of the DART source through the addition of a custom gas-ion separator tube (GIST; IonSense Inc,. Saugus, MA, USA) connected to a Vacuubrand 2 C diaphragm pump (Vacuubrand, Wertheim, Germany). The DART source was operated with helium at 1 L min–1 at a gas temperature of 100–400 °C, discharge voltage of 3500 V and a grid electrode voltage of 50 V. The particular flow rate used in this study was chosen because it is the maximum allowed by the mass spectrometer. The interface includes a pre-pumped gas ion separator tube (GIST) to reduce the vacuum load on the first differentially pumped region of the instrument. However, even a small rise in helium flow rate (to 1.2 L min–1) raises that stage’s pressure beyond the safe operating conditions and automatically shuts down the power supplies providing the ion optics potentials. The mass spectrometer settings were as follows: end plate offset –500 V, capillary –2000 V, dry gas (nitrogen) 2 L min–1, dry gas temperature 150 °C, spectra acquired at 1 Hz in the 50–1000 m/z range.
2.3 Experimental Set-up
3 Results and Discussion
3.1 Sampling Region Temperature Gradient
3.2 Spatial Sensitivity and Dynamic Range
The sensitivity map differed at higher sampled concentrations (500 μM) with the highest sensitivity measured at position B1 (Fig. 3c, f, and i). These sensitivity maps corresponded well with the observed temperature gradients (Fig. 2) and suggest that dilution from the DART gas was not as significant of an effect. At such high concentration levels, sensitivity more closely follows the relative rates of thermal desorption throughout the sampling grid. Sensitivity changes were more pronounced at lower temperatures where the increase in relative intensity at B1 compared with B2 was 80% and 100% for DART gas temperatures of 200 and 300 °C, respectively (Fig. 3c and f). At these two settings, the temperature drop-off causes the effective gas temperature to become much closer to the DMMP BP (181 °C), reducing the thermal desorption rate. At a temperature setting of 400 °C, the increase in intensity from B1 to B2 was only 40% due to the temperature still being much higher along the B sampling column (Fig. 3i).
It is also important to point out that sensitivities were ~80%–90 % lower in any row along columns A and C compared with column B. The two major factors causing the low sensitivity were the lower overall temperatures at these positions and poor ion transmission trajectories towards the inlet. Temperatures were lower by ~60%–80 % on the outer columns A and C compared with column B (Fig. 2). Also, the sample positions along columns A and C were off axis to the GIST inlet. If ionized, the low quantity of desorbed molecules would have a more indirect and longer trajectory to the inlet. A possible means to further improve ion sampling would be to use a flared capillary inlet with a large entrance aperture at the opening of the GIST tube.
Following these experiments, DMMP was sampled in all three positions (B1, B2, B3) and gas temperatures (200, 300, and 400 °C) over a wider concentration range (25 μM to 1 mM) to assess the available dynamic range (Supplementary Figure S2). As a whole, three decades of concentration were detected with corresponding increases in signal response. Concentrations above 1 mM were not used due to laboratory safety reasons although judging from the observe intensities, concentrations near 10–25 mM should be detected with similar trends. Throughout all tested variables, there was a decrease in overall sensitivity with an increase in set DART gas temperature (Supplementary Figure S2). This alludes to carefully tuning the rate of desorption to be sufficient enough to evaporate excess solvent (water), but not too high to induce fragmentation and/or produce analyte neutrals faster than the reaction with protonated reactive species. However, this may be a minor effect that compounds with a lower volatility, such as higher molecular weight molecules, which may not experience due to the requirement for additional heat to be thermally desorbed.
Overall the B3 position corresponded to the poorest overall sensitivity at any tested concentration level due to poor thermal desorption. However, this position provided the highest linearity. The most discernable changes in signal intensity with respect to concentration was observed for B3 (Supplementary Figure S2a). Extension of the sampled concentrations to probe the linear dynamic range (1–100 μM) showed strong linearity at B3 at all three temperatures (200 °C: r2 = 0.981, 300 °C: r2 = 0.975, and 400 °C: r2 = 0.985) (Supplementary Figure S3a). At lower concentrations (25, 50, 75, and 100 μM), the middle position, B2 had the highest sensitivity supporting the previous results (Supplementary Figure S2b). The linear dynamic range for B2 at 200, 300, and 400 °C was lower than at B3 with r2 = 0.940, 0.876, and 0.880, respectively (Supplementary Figure S3b). Position B1 also corresponded well to the previous sensitivity map showing the highest sensitivity for the highest tested concentrations (250 μM, 500 μM, and 1 mM, Supplementary Figure S2c). The linear dynamic range for 200, 300, and 400 °C was lower than at B3 with r2 = 0.911, 0.781, and 0.980, respectively (Supplementary Figure S3c). All these data suggest that future routine sensitive quantitative or semiquantitative DART methods would likely see advantageous results by positioning samples in regions where both the role of suction to improve ion transmission and localized heating are maximized.
3.3 Ion Suppression
Oddly, for all three temperatures, there was a dip in the level of suppression at the equimolar concentration of 100 μM. In most trials, this dip was small and within the experimental error, however position B1 suffered from the largest dips. This effect cannot be explained by ion transmission or thermal dissipation effects since transmission losses should be less noticeable at higher concentrations, and because position B1 experiences the highest temperatures (Fig. 2). The dip may be attributed to fluctuations in reactive species under those conditions. For clusters with n = 7–13, there was a slight increase in intensity for position B1 at 50 μM compared with 100 μM (Supplementary Figure S3a and b). The extreme dip at 300 °C may have been caused from a gas turbulence disturbance between trials.
One other interesting, but currently unexplained, phenomenon was the relative higher intensity of midsized water clusters at n = 9, 11, 13, and 17 in some trials (Supplementary Figure S3). As a whole, the number of stable water cluster isomers increases exponentially with n . The higher abundance of specific clusters has been observed previously with DART , but that study showed relative higher levels of water clusters n = 3, 6, 9, and 12 for various temperatures (175, 250, and 325 °C) and flow rates (2, 4, and 6 L min–1). Although there are no significant differences in binding energies  and bond dissociation energies  for water clusters in the range 9 ≤ n ≤ 17, the atmospheric pressure interface in the instrument used in this study (GIST-capillary-dual ion funnel) is much different than that in previous work (cone/skimmer orifice) . Therefore, we believe that the observed differences in water cluster species are an effect caused more by the ion transfer optics design and reduced pressure region architecture than the by preferential formation or reactivity of particular species.
After observing the major contributing factor to ion suppression was differences in PA rather than BP, conditions to reduce this effect were explored. The same analytes (p-anisidine and isoquinoline) were used in these tests, but molar ratios were varied from 1 (50 μM each of p-anisidine and isoquinoline), to 10 (500 μM of p-anisidine and 50 μM isoquinoline), such that the lower PA analyte would have an increased proportion of neutrals in the ionization region. These competition experiments were further compounded by changing the position where each individual analyte was simultaneously introduced to investigate how the relative increase of neutrals would be affected by ionization reactions in space.
A similar trend was observed when the positions were reversed such that the higher PA isoquinoline sample was continuously infused at B1 or B2 and the lower PA p-anisidine was injected at B3. Ion suppression levels were much higher in this scenario, because the higher PA molecule was closer to the DART gas exit where it would undergo the majority of reactions with protonated water clusters and scavenge protons from any p-anisidine protonated molecules in transit to the mass spectrometer inlet. When isoquinoline was at B2 and p-anisidine was at B3, a remarkably high suppression level of 4543.0× ± 1406.0 was observed at an equimolar ratio (green line, Fig. 6). As the molar ratios increased to 2, 5, and 10, the ion suppression decreased to 107.8× ± 50.4, 73.1× ± 6.1, and 19.4× ± 2.2, respectively. The levels of suppression were further decreased when the isoquinoline sampling position was moved closer to the DART source (B1) while p-anisidine remained at B3, thereby increasing the distance between where the two molecules were introduced. For molar ratios of 1, 2, 5, and 10 ion suppression was 255.6× ± 13.3, 147.8× ± 7.5, 71.6× ± 3.7, and 8.8× ± 1.2, respectively (blue line, Fig. 6). In this sampling configuration, there was a higher likelihood that isoquinoline would ionize from reactions with protonated water clusters and not from proton scavenging. Nevertheless, there would always be suppression in the concentration range tested since isoquinoline was closer to the ionization source.
Spatial sensitivity and ion suppression effects are often overlooked in applications involving ambient desorption/ionization techniques, however the results presented here demonstrate the magnitude that these can reach in certain sampling conditions. Although temperature gradients play a role in thermal desorption, concentration-dependent ion yield “hot-spots” where sensitivity was optimal may not coincide with the hottest regions in space. Additionally, differential analyte volatility plays a smaller role in ion suppression than differences in PAs. Suppression due to different PAs may be exacerbated by the spatial location of sampling. Although the experiments outlined here used continuous infusion of liquids to acquire steady-state signals, it is expected that solid samples placed within the ionization region for rapid screening purposes would show similar, if not more extensive, ion suppression due to additional flow instabilities and induced mixing in the ionization region. Future investigations of ion suppression in DART will incorporate multicomponent mixtures and higher molecular weight compounds to determine if the mass of the analytes affects ion suppression.
The authors give special thanks to Sam Mize at the College of Science machine shop for the sample block construction. The authors acknowledge support for this study by an NSF CAREER award to F.M.F.
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