Identification and quantification of VOCs by proton transfer reaction time of flight mass spectrometry: An experimental workflow for the optimization of specificity, sensitivity, and accuracy

Abstract Proton transfer reaction time of flight mass spectrometry (PTR‐ToF‐MS) is a direct injection MS technique, allowing for the sensitive and real‐time detection, identification, and quantification of volatile organic compounds. When aiming to employ PTR‐ToF‐MS for targeted volatile organic compound analysis, some methodological questions must be addressed, such as the need to correctly identify product ions, or evaluating the quantitation accuracy. This work proposes a workflow for PTR‐ToF‐MS method development, addressing the main issues affecting the reliable identification and quantification of target compounds. We determined the fragmentation patterns of 13 selected compounds (aldehydes, fatty acids, phenols). Experiments were conducted under breath‐relevant conditions (100% humid air), and within an extended range of reduced electric field values (E/N = 48–144 Td), obtained by changing drift tube voltage. Reactivity was inspected using H3O+, NO+, and O2 + as primary ions. The results show that a relatively low (<90 Td) E/N often permits to reduce fragmentation enhancing sensitivity and identification capabilities, particularly in the case of aldehydes using NO+, where a 4‐fold increase in sensitivity is obtained by means of drift voltage reduction. We developed a novel calibration methodology, relying on diffusion tubes used as gravimetric standards. For each of the tested compounds, it was possible to define suitable conditions whereby experimental error, defined as difference between gravimetric measurements and calculated concentrations, was 8% or lower.

largely dependent upon reduced electric field within the drift tube. This parameter is conveniently summarized by the E/N parameter, which can be easily modified by changing drift voltage, temperature, or pressure. Lower E/N values will result in a reduction in drift velocity and fragmentation, with increased sensitivity. Under the ionization conditions most commonly employed in PTR-MS (E/N = 120-150 Td, 1 Td = 10 −17 V cm 2 ), high fragmentation can sometimes compromise the detection and identification of some compounds. This is well exemplified by aldehydes: when using H 3 O + as primary ion, dissociative reaction channels prevail and the relative abundance of the protonated ion [M + H] + is as low as 6% to 9% with C5-C8 compounds. 5 To date, even though the potential benefits of working under conditions of low reduced drift field are theoretically known, the actual exploration of such conditions is limited. The reactivity of several VOCs (mostly alkanes) was studied employing NO + as primary ion at 60 Td E/N. 6 The possibility of employing similar conditions for H 3 O + was demonstrated, provided cluster chemistry and adduct formation are taken into account. 7 The use of O 2 + as primary ion under low reduced drift field conditions has, to our knowledge, never been reported.
Several techniques are nowadays available for calibration of a VOC measurement. The most commonly adopted approach relies on gas calibration mixtures. 8 The main limitation lies in the cost and reduced availability of VOCs as diluted calibration gases. The use of dynamic solution injection (DSI) obviates the problem of sample availability: in DSI, a solution containing the compounds of interest is vaporized into a heated chamber and injected into the instrument. 9 The main disadvantage of DSI lies in the need to find a suitable medium where to dissolve the analytes of interest: water is ideally suited for PTR-MS analysis, but it will not dissolve nonpolar VOCs, whereas the use of most organic solvents or co-solvents will saturate the detector. Permeation or diffusion tubes represent the most straightforward approach 10 : a permeation/diffusion device can be prepared using any available compound, regardless of solubility. The VOC concentration in the gas phase can be varied by altering the device equilibration temperature and/or flow. Most importantly, by measuring analyte losses at the end of the experiment, it is possible to standardize the results against a gravimetric measurement.
The aim of the work presented in this manuscript was to describe an experimental workflow that helps researchers to set up the appropriate methods for optimum sensitivity, specificity, and accuracy of VOC measurements. The 13 compounds chosen for this study belong to 3 chemical classes (aldehydes, fatty acids, and phenols). Fragmentation patterns were determined at different E/N values under breath-relevant conditions. The impact of water vapor on fragmentation was assessed, and analytical accuracy was determined by comparison with gravimetric standards. The compounds examined in the work were previously found to be elevated in the exhaled breath of patients suffering from oesophago-gastric adenocarcinoma. 11 These results were obtained using another established technique for VOC analysis: selected ion flow tube mass spectrometry (SIFT-MS). 12 The present work also provides interesting insight regarding differences and analogies between PTR-MS and SIFT-MS chemistry.

| Fragmentation pattern determination
For the determination of the fragmentation patterns, we adapted a procedure described in the literature. 13 Individual VOC solutions were prepared in de-ionized water at concentrations ranging from 0.03 to 1% (v/v) for the aldehydes and fatty acids and at 100 mg/L for the phenols.
A 500-mL glass bottle, equipped with a Drechsel head, was filled with 100-mL de-ionized water and kept in a water bath at 37°C. All raw signals were modified correcting for the detector-specific mass discrimination properties, by establishing the so-called "transmission curve." This was obtained by measuring a certified mixture of aromatic hydrocarbons, all at the concentration of 100 ppbV (TO-14 Aromatics Subset Mix, Sigma Aldrich). The transmission values were established by interpolating the measured intensity values of reference mass peaks by means of a natural spline. Reaction rate constants were calculated using Su's parametrization approach. 15 This allows to consider non-thermal conditions typical of commercial PTR-MS instruments, allowing for more accurate quantification. 16 All reaction rate constants and the parameters used to calculate them are reported in

| Data analysis
Data were extracted using PTRMS viewer version 3.2.2.2 (Ionicon Analytik). Additional data analysis was conducted using in-house generated scripts written using R programming language. 17 3 | RESULTS AND DISCUSSION 3.1 | Aldehyde reactivity using NO + as primary ion  (Table 1), and analogous behavior is observed for butanoic and pentanoic acid. Figure 1 shows how at E/N = 84 Td, the best compromise conditions were obtained, with minimized adduct and fragment formation and 65% to 73% protonated ion relative abundance. Similarities were observed between PTR-MS and SIFT-MS chemistry with the latter also being characterized by protonation and water removal as preferred reaction channels for butanoic and pentanoic acid. 21 The perusal of an up-to-date SIFT-MS Profile 3 instrument kinetic library (unpublished results) reveals that water adducts are also encountered among the products of the reaction between H 3 O + and fatty acids.  (Table 1). It is also worth mentioning that, when using a O 2 + as primary ion, a product with mass M + 1 is observed with the 3 phenols; this can derive from the 13 C isotopologue of the M + ion, but its relative intensity exceeds the expected abundance of 13 C. It can be hypothesized that this mass peak comes from the overlap of 2 reaction products: the 13 C isotopologue and a protonated ion. The latter could be originating from a ligand switching reaction from a water cluster, taking place when water vapour is present within the drift tube. This is followed by a dissociative collision, made possible by the presence of a neutral species M (eg, N 2 ) acting as a third body (possible reaction mechanism reported for ethyl-phenol in Table 2). A similar mechanism mixed ionization mode. 23 The presence of this reaction channel must be considered when using O 2 + to measure phenols at low E/N values in water-rich matrices; its contribution is estimated as follows: where the relative abundance of the protonated ion is obtained by subtracting the expected contribution of the 13

| Other compound/primary ion combinations
The study of the ionization of aldehydes with H 3 O + and O 2 + and fatty acids with NO + and O 2 + as primary ions provides interesting insight on

| Effect of drift voltage on sensitivity and comparison between dry and humid conditions
The choice of the optimal conditions in terms of E/N should aim to the generation of a single product ion from each compound/primary ion combination. In order to increase the informational content of the mass spectrum and to minimize overlaps, the product ion should ideally be a molecular ion generated by charge transfer, a quasi-molecular ion originating from hydride abstraction reactions, or a protonated ion.
Conditions of reduced drift voltage result in increased sensitivity, thanks to the reduced fragmentation, but also due to the increase in  the switch from humid to dry air resulted in no effect for nonanal and decanal, while for shorter chain aldehydes, a moderate but measurable increase in fragmentation was observed (up to 9% with butanal). The switch from humid to dry air showed no measurable impact on fatty acids (H 3 O + as primary ion) and phenols (NO + as primary ion).

| Comparison with a gravimetric measurement
The  Table 2). The procedure showed good repeatability, as coefficients of variability in the assessment of diffusion rates were below 7%. The proposed protocol provides accuracy values similar to those reported using gas calibration mixtures, 6 with the advantage of being applicable to any compound for which a pure standard is available.

| CONCLUSION
This work outlines an experimental workflow for the reliable detection and quantification of target compounds by PTR-MS. Based on this original workflow, we recommend researchers to approach the problem by performing a thorough screening of reduced drift field conditions with different primary ions, also testing E/N value below 120 Td, which are in most cases neglected. As demonstrated by our results, this strategy allowed to define an optimal range of reduced electric field (E/N = 72-96 Td) for the detection and quantification of aliphatic aldehydes, with a 3-fold increase in sensitivity compared with conditions reported in the literature. 5,18 Quite interestingly, the use of low reduced drift field also disclosed surprising similarities between PTR-MS and SIFT-MS chemistry. SIFT-MS is known to afford lower fragmentation than PTR-MS due its less energetic ionization conditions: the findings reported in this work suggest that, by means of the choice of appropriate ionization settings, suitable conditions can be found where the wealth of information available on SIFT-MS fragmentation patterns of VOCs (the so-called "kinetic libraries") might also be used to provide useful indications for the interpretation of PTR-MS spectra.
As additional step of the workflow, we suggest evaluating the impact of a change in humidity on VOC fragmentation patterns. This experiment is of particular interest for those fields of research (eg, breath analysis, atmospheric chemistry) where a variation in relative humidity of the matrix can affect the results. Finally, we provide a set of all-purpose experimental conditions for gravimetric calibration, providing a quantitation accuracy within 8% of the reference standard.
The use of gravimetric calibration provides an undeniable advantage over other available methods (eg, gas calibration mixtures and DSI) as it is applicable to any VOC that is readily available as authentic standard.