Glow flow ionization mass spectrometry of small molecules. A comparison of a glow flow ionization source (‘GlowFlow’) with electrospray ionization and atmospheric pressure chemical ionization

Rationale Ionization by atmospheric pressure gas discharge has been employed for a long time in mass spectrometry. Inductively coupled plasma mass spectrometry is an exemplar, and widely used for elemental analysis. The technique has less uptake in organic mass spectrometry. We describe a simple source design that can be readily implemented in most atmospheric pressure ionization (API) systems and compare its performance with that of electrospray ionization (ESI) and atmospheric pressure chemical ionization (APCI). Methods An in‐house designed helium gas discharge source (referred to as ‘GlowFlow’) was used on a Xevo G2‐S time‐of‐flight mass spectrometer. The GlowFlow source was transferred to a compatible Xevo TQ‐S triple‐quadrupole mass spectrometer using an ultrahigh‐performance liquid chromatograph inlet. Its performance was compared to that of Waters ESI and APCI sources. Results Preliminary results of GlowFlow on the Swansea instrument are presented to establish context and include analysis of low‐molecular‐mass polymers, benzoic acid and cinnamic acid. Comparison of performance on the Xevo TQ‐S triple‐quadrupole mass spectrometer involved three test mixtures. The method limits of detection (six‐mix) for positive‐ion GlowFlow source were between 0.03 and 10.00 pg with good linear response over two to four orders of magnitude and values of R 2 > 0.98. The GlowFlow ionization source provided a signal intensity that was an order of magnitude greater than that of ESI for an atmospheric pressure gas chromatography standard mix and ionized several compounds that ESI could not. Conclusions The current GlowFlow design is relatively simple to retrofit to most API systems due to its small size. The sensitivity of the GlowFlow design is typically an order of magnitude less than that of ESI in positive‐ion mode, but similar in sensitivity in negative‐ion mode and comparable to that of APCI.


| INTRODUCTION
Glow flow ionization (GFI) is a term describing atmospheric pressure ionization (API) based on a gas discharge where helium is typically used. We propose the acronym 'GFI' can be used to define an array of similar techniques (some discussed below) used in analytical mass spectrometry. A recent GFI design from our group, 1,2 which we refer to as 'GlowFlow', incorporates a compact and simple construction that is suitable for analytical mass spectrometry. This GFI source can be readily retrofitted to most API systems with minimal modifications.
A comparison study was undertaken, using a Xevo TQ-S triplequadrupole mass spectrometer based at the Waters' research group (Waters Corp., Wilmslow, UK), between our GlowFlow source, an electrospray ionization (ESI) source and an atmospheric pressure chemical ionization (APCI) source.
Our earliest GFI designs followed the work of Hieftje and coworkers 3 (flowing atmospheric pressure afterglow, FAPA), where a remote discharge cell feeds a helium afterglow onto the sample, in a gaseous, liquid or solid form. 4 At that time our laboratory ran thousands of non-polar samples every year by electron ionization and atmospheric solids analysis probe. 5 Thus, we were motivated to search for techniques that could handle compounds, from non-polar to polar chemistries, using a single method. Atmospheric pressure glow discharge (APGD) ionization seemed a prospect to study for that venture. The GlowFlow design is simple and can analyse wide ranging chemistries whilst displaying good analytical figures-of-merit, particularly in negative-ion mode.
Ionization mechanisms occurring in APGD have been described previously 6 1,6 In the literature, there have been several gas discharge techniques reported, often using argon gas and with some aimed at lightweight portable mass spectrometry applications. Of note in the literature are those for surface analysis, 7 analytical mass spectrometry, 8 microplasma-based FAPA, 9 a mini-FAPA source coupled to capillary electrophoresis, 10 differentiation of functional isomers 11 and the helium plasma ionization designs of Pavlov et al. 12,13 The latter design seems very promising for GFI applications as it incorporates a dopant bleed into the analyte flow providing inter alia a source of protons and reagent species to enhance ionization. These sources are based on APGD where the analyte is directly exposed to the glow discharge region. In contrast, the direct analysis in real time source 14 uses a helium, argon or nitrogen corona discharge in combination with an exit electrode that is biased to remove electrons and negative ions, or alternatively positive ions, from the discharge, whilst preserving the metastable neutral species. The GlowFlow source, operated in positive-ion mode, has consistently given weaker signal than electrospray in earlier measurements at Swansea. This suggests a deficit of proton-donating reagents (e.g. H 3 O + ) which could arise as the nebulizer gas and GFI source, in that instrument, employ pure nitrogen and helium, and thus the source is operating in an environment deprived of protons. A similar dopant approach has been used successfully in atmospheric pressure photoionization sources. 15,16 In recent GFI studies we examined a range of sample types that involved several ionization mechanisms. 1,2 Here we briefly introduce four further GFI examples for context: polymers, LC/MS studies, benzoic acids and atmospheric pressure gas chromatography (APGC) standards; however, the main thrust of the article is the comparison of GlowFlow with ESI and APCI on a triple-quadrupole instrument. A Xevo TQ-S mass spectrometer was chosen as our GlowFlow source could be directly fitted to the triple-quadrupole system as the housing, mechanical, gas and electrical connections are all common. 17 The study was carried out on well-established test mixtures in positiveand negative-ion modes. Ultrahigh-pressure liquid chromatography data were also obtained to verify the chromatographic fidelity of the GlowFlow arrangement (but not included herein). The samples were delivered via an integrated APCI IonSABRE II (heated nebulizer) probe which was positioned perpendicular to the GFI source and mass spectrometer inlet, as shown in Figure 1.
The addition of 'dopants' to enhance ionization is well documented: from prior sample derivatization in electron ionization, to more recent examples where the dopant is added directly to the ionization region such as in atmospheric pressure photoionization 15,16 and the extensive work of Trimpin. 17 The GlowFlow study using the triple quadrupole did not include dopant studies, due to limited time.

| Analysis of low-molecular-mass polymers by GlowFlow
Three low-average-molecular-mass polymers, namely PEG 400, PPG 725 and PEI, were selected for analysis using GFI mass spectrometry on the Xevo G2-S instrument in full-scan mode using the GlowFlow source. The samples were prepared at concentrations of 1 μg μL À1 , and 1 μL was syringed on the end of a glass capillary before being introduced into the source using a solids analysis probe. Intense

| Negative GlowFlow mass spectrometry of benzoic and cinnamic acids
Negative-ion mass spectrometry is not as commonly used as the positive-ion counterpart, because fewer compound classes undergo the process to form negative ions, such as deprotonation.
F I G U R E 1 (A) Image of the GlowFlow system in a Waters Xevo G2-S universal source housing. The ionization source is to the right, and axially opposite the mass spectrometer entrance (left

| Analysis of 4-fluorobenzoic acid by loopinjection GlowFlow
The compound 4-fluorobenzoic acid was selected and analysed at a concentration of 100 pmol μL À1 by loop injection using a 1 μL

| Comparison of the GlowFlow source with ESI
Analysis of the 'six-mix solution' containing acetaminophen, caffeine, sulfadimethoxine, hydroxyprogesterone and verapamil was undertaken using the GlowFlow ionization source on an Acquity HPLC coupled to the Xevo TQ-S triple-quadrupole mass spectrometer. [22] The GlowFlow source was operated in two   (Figures 6 and 7) and a summary of the data is presented in Tables 2 and 3 Figure S1). The GlowFlow ionization source is at least an order of magnitude less sensitive compared to the standard ESI method for these specific compounds.

| Comparison of ionization sources for the detection of extractables and leachables (E&L) 21
Analysis of 18 compounds which are commonly detected as E&L in plastics was undertaken with an Acquity HPLC coupled to the Xevo T A B L E 2 Summary of the results for six-mix compounds using the GlowFlow ionization source in combination with the APCI probe. Listed are the linear dynamic range (1 d.p.), the coefficient of variability (R 2 ), the lowest detectable amount on column, the corresponding standard deviation (SD) at the lowest amount on column and the calculated instrument detection limit (IDL)  ESI data. The results for positive-ion mode are shown in Figure 8. The GlowFlow and APCI sources both showed reduced sensitivity with respect to ESI. As is observed with the six-mix compounds, the glow flow source is typically an order of magnitude lower in sensitivity than ESI (positive ion) under these experimental conditions. However, the data for the protonated molecules are not necessarily representative for all the compounds studied. As can be seen in Figure 9, the protonated molecule is not discernible in the mass spectrum of

| Analysis of APGC standard by direct infusion
The APGC standard containing eight compounds (see Table S2) was analysed using the direct infusion method at a flow rate of 20 μL min À1 and the compounds were initially detected in full-scan mode

| CONCLUSIONS
GFI appears to be a versatile ionization technique, with potentially high sensitivity in some applications. The technique is currently not widely available and its potential sensitivity and range of application are generally unknown in the field of organic mass spectroscopy. It is generally an easy technique to fit to API systems and the GlowFlow design is particularly simple to retrofit due to its small size. GFI's sensitivity is in the picomole to femtomole range; however, we intend to further research the GlowFlow design with a dopant bleed and hope to approach the sensitivities exhibited by an ESI source. The GlowFlow design exhibits a fast response making it compatible with ultrahigh-performance liquid chromatography. GFI in broad terms has considerable potential for development with small-molecule work (under around 800 Da). Due to its small size, it can be mounted and used alongside other API sources and thus be readily switched in and out of operation without source removal and provide added value to an analyst's tools. There have been only limited studies with higher mass compounds since it is currently insensitive at higher mass.
These data have shown GlowFlow ionization to be an order of magnitude less sensitive than ESI in positive-ion mode, but nevertheless, it does exhibit a limit of detection in the low nanogram to femtogram range, with values of R 2 > 0.98 over two to four orders of magnitude. The GlowFlow ionization source provided signal intensity of an order of magnitude greater than that of ESI for the APGC standard mix and ionized several compounds the ESI could not.
The high and positive Log P values for the APGC compounds (Table S2) suggest that GlowFlow is particularly suited for the analysis of more non-polar compounds, which is consistent with historical observations of ionization sources that are based on discharges. 6