As the APCI-MS for 1 and 3 both showed loss of ketene from the molecular ion but 1 also showed a loss from an intermediate fragment, we sought to further explore this possibility using DFT calculations. This would allow a direct comparison of activation energy barriers between the known ketene producer 1 and vitamin E acetate. The energy profiles and transition states (TS) for the elimination of ketene from 1 , 3 and 6 were calculated in the gas phase at the M06-2X/6–311G (d,p) level using the Gaussian 09 package ( 18 ).

Positive-mode APCI-MS of (A) phenyl acetate 3 with source voltage of 15 eV showing loss of ketene; (B) vitamin E acetate 1 with source voltage of 20 eV showing loss of ketene from both the parent ion and a fragmentation species of m/z 207.2; (C) vitamin E 2 with source voltage of 20 eV with sole fragmentation to [Ar 3 OH 2 ] + .

Phenyl acetate 3 was analyzed using atmospheric pressure chemical ionization (APCI) mass spectrometry (MS) at a series of increasing source cone voltages (CVs) from 5 to 30 eV. At 15 eV the parent ion [M+H] + was identifiable as the mass-to-charge ratio (m/z) of 137.1 (10%) with fragmentation ion at 95.2 (100%) ( Fig. 2A and SI Appendix, Fig. S1 ). This corresponds with a mass loss of 42 from the parent ion, which is equivalent to ketene 4 and consistent with previous studies that used EI MS instrumentation ( Fig. 3A ) ( 12 ). An identical analysis of vitamin E acetate 1 also showed the elimination of ketene (m/z = 42) from the parent ion (as seen for 3 ) and from a fragment with m/z of 207.2 ( Fig. 2B and SI Appendix, Fig. S2 ). A representative spectrum taken with CV of 20 eV shown in Fig. 2B reveals the parent ion m/z of 473.6 (100%) with fragmentation ions at 431.5 (15%), 207.2 (65%), and 165.2 being the most prominent features. Increasing the CV to 25 eV decreased the abundance of the parent ion and increased the fragmentation such that (m/z) abundances were 473.6 (10%), 431.5 (10%), 207.2 (100%), and 165.2 (10%) ( SI Appendix, Fig. S2 ). Clearly two fragmentation patterns can occur for 1 with fragmentation pattern A due to elimination of ketene 4 from the molecular ion producing vitamin E 2 ([Ar 1 OH 2 ] + , m/z 431.5) whereas fragmentation pattern B has loss of prist-1-ene 7 producing 6 ([Ar 2 OH 2 ] + , m/z 207.2) from which elimination of 4 generates [Ar 3 OH 2 ] + (m/z 165.2) ( Fig. 3B ). As a control, vitamin E 2 was also included in the MS study showing, as expected, no m/z loss of 42 and showing the same [Ar 3 OH 2 ] + (m/z 165.2) species observed as the lowest molecular weight fragment from 1 and attributable to structure 8 ( Fig. 2C and SI Appendix, Fig. S3 ). As both 1 and 3 showed the same MS fragmentation pattern of ketene elimination, this provides the chemical basis by which the pyrolysis of vitamin E acetate would produce ketene.

As two pathways are operable for the elimination of ketene from 1, DFT calculations were carried out to determine if either one was more favorable. The competitive first steps of the two pathways are either loss of ketene from 1 forming 2 or loss of prist-1-ene 7 from 1 forming 6 , from which subsequent elimination of ketene would occur ( Fig. 3B ). Conversion of 1 into 6 + 7 is akin to the pyrolysis of a chroman ring. Chroman pyrolysis occurs through a retro Diels–Alder reaction to produce ortho-quinone methide and ethane by C–C and C–O bond cleavage in the dihydropyran ring ( 21 ). Employing gas-phase M06-2X/6–311G (d,p) calculations, the TS energy barrier for the formation of 6 from 1 was determined to be 58.8 kcal/mol ( SI Appendix, Fig. S7 ). This value is lower than conversion of 1 into 4 + 2 by 10.7 kcal/mol. As such, it seems probable that both ketene-producing pathways may be in operation, i.e., 1 → 4 + 2 and 1 → 6 → 4 + 8 but that the latter is more favorable.

Optimized geometries [M06-2X/6–311G(d,p) level] for the TS corresponding to the pyrolysis of phenyl acetate 3 (Left) and vitamin E acetate 1 (Middle) and 6 (Right), leading to the elimination of ketene. Simplified structures used for calculation of 1 and 2 without C 15 H 32 alkyl group. For details of calculation data see SI Appendix, Figs. S4–S6 .

Gas-phase M06-2X/6–311G (d,p) calculations from 3 gave an activation energy of 65.4 kcal/mol via this pathway, confirming that it would be energetically feasible under pyrolysis conditions ( Fig. 4 , black energy profile). At this level of theory, the barrier for ketene elimination from 1 was only 4.1 kcal/mol higher than 3 at 69.5 kcal/mol, indicating comparable pyrolysis reactivity for both ( Fig. 4 , blue energy profile). As the MS fragmentation data for 1 indicated ketene elimination from fragment 6 can also occur, its energy barrier to release ketene was calculated and found to be 65.9 kcal/mol, which is lower than 1 and comparable with 3 ( Fig. 4 , green energy profile). The optimized geometry of the four-membered cyclic TS for the unimolecular decomposition of 1 , 3 , and 6 showed similarity for each and that ortho-methyl substituents of 1 and 6 acetate did not impede ketene elimination ( Fig. 5 and SI Appendix, Figs. S4–S6 ). Additionally, intrinsic reaction coordinate calculations confirmed the TS structures associated with the products and reactants along the minimum energy pathway.

Calculated energy profiles for the pyrolysis of the three aryl acetate substrates 1 , 3 , and 6 via concerted [1,3] hydrogen shift mechanism. Relative free energies (kcal mol −1 ) for starting materials, TS, and products formed for the pyrolysis of phenyl acetate, vitamin E acetate, and 6 leading to the elimination of ketene 4 . For details of calculation data see SI Appendix, Figs. S4–S6 . Simplified structures used for calculation of 1 and 2 without C 15 H 32 alkyl group.

Experimental Analysis of Vaped 1.

With experimentation and theoretical evidence for the elimination of ketene from 1, we felt compelled to directly investigate the effect, if any, on vaping vitamin E acetate. However, it should be noted that these experiments were not designed to exactly replicate a user’s experience; rather, the goal was to determine the vaping pyrolysis effect on 1 as a single pure substance at the chemistry molecular level. Extrapolation of the exact relevance of these results to the direct cause of lung injury is beyond the scope of this preliminary report due to the diversity in vaping devices, mixtures, and their modes of use. As described in Methods, the isolated mixture of vaped 1 was collected and analyzed as either the total vaped mixture or following separation into its volatile (VC) and nonvolatile components (NVC). APCI-MS analysis was inconclusive, as spectra closely resembled that of pure 1, although it was suspected that changes may be masked by the fragmentation pattern of remaining 1 and the formation of hydrocarbon fragments that are not APCI-MS responsive (SI Appendix, Fig. S8). As such, NMR data were collected for the total vaped material and individually for the VC and NVC of the vaped mixture. Comparison of the 1H NMR spectrum of the entire vaped material with that of pure 1 was revealing as it showed that significant chemical transformations had occurred. Numerous peaks with complex splitting patterns, not observed for vitamin E acetate, between 4.6 and 7.4 ppm were recorded in the vaped material and the spectral region between 0.8 and 2.1 ppm showed increased complexity (Fig. 6 and SI Appendix, Fig. S9).

Fig. 6. (Top) 1H NMR spectrum of pure 1. (Bottom) 1H NMR spectrum of entire isolated vaped mixture from 1. For clarity the peak at 7.36 ppm is not shown in cropped view, see SI Appendix, Fig. S9 for full spectra. Inset box (i) indicates VC; box (ii) indicates mixture of VC and NVC; box (iii) primarily NVC.

Separate 1H NMR spectra were obtained for both NVC and VC. Close examination of the NVC spectrum and comparison with 1 and 2 provided important insights (Fig. 7 and SI Appendix, Fig. S10). This showed that NVC comprised vitamin E acetate 1 (key singlet peaks at 2.09, 2.03, 1.98 ppm), 2,3,5,6-tetramethylcyclohexa-2,5-diene-1,4-dione, (duroquinone) 8 (key singlet peak at 2.01 ppm), and prist-1-ene 7 (key doublet peak at 4.68 ppm). The assignments of 7 and 8 were further confirmed by 13C spectra, proton-carbon correlations using heteronuclear single quantum coherence spectroscopy, and heteronuclear multiple bond correlations (SI Appendix, Fig. S11). Comparison with a spectrum of pure 2 showed that, perhaps surprisingly, no vitamin E was contained in the mixture (key singlet peaks at 2.18, 2.13 ppm, two overlapping singlets) (Fig. 7). Integration of a methyl singlet of 1 versus that for duroquinone 8 gave an estimate of vape-induced pyrolysis between 15–20%. This amount of pyrolytic conversion was consistent across six independent experiments. The formation of 8 was confirmed by high-performance liquid chromatography (HPLC) and gas chromatography (GC)-MS experiments (SI Appendix, Figs. S12 and S13).

Fig. 7. Portion of the 1H NMR spectrum of vitamin E acetate 1, vitamin E 2, and the isolated NVC of vaped mixture. A red asterisk marks the aryl methyl signal for duroquinone 8 (full spectra in SI Appendix, Fig. S10).

NMR analysis identified the VC in the mixture as benzene (key singlet peak at 7.36 ppm), butadiene (key multiplet peak at 6.36 ppm), propene (key d,d,q peaks at 5.83 ppm), ethene (key singlet peak at 5.41 ppm), 2-methylprop-1-ene (key singlet peak at 4.66 ppm), and lower molecular weight 1-methyl-1-alkyl-alkenes (key doublet peak at 4.67 ppm) (Fig. 8). Trace amounts of tetrahydrofuran, formaldehyde, and short-chain aliphatic aldehydes were also observed (SI Appendix, Fig. S14). Propene was the most abundant volatile material with a three- to fourfold stoichiometric ratio greater than that of duroquinone 8. This indicates that upon release of prist-1-ene 7 from 1, it fragments into numerous propene units and the other identified alkenes. Trace aldehydes would be oxidation products of these alkenes and benzene a downstream byproduct from them.

Fig. 8. Portion of the 1H NMR spectrum of the isolated VC following vaping vitamin E acetate 1. See SI Appendix, Fig. S14 for full spectrum including benzene peak at 7.36 ppm. Red asterisk, 1-methyl-1-alkyl alkenes; blue asterisk, propene; black asterisk, butadiene.

The routes to forming the spectrum of compounds generated by the vape pyrolysis of 1 are illustrated in Fig. 9. In pathway A, ketene is released from 1 to produce vitamin E which undergoes C–C and C–O bond breaking of the dihydropyran ring to produce 8 and 7. Pathway B generates the same mixture of end products but by a different sequence such that first the dihydropyran ring cleaves followed subsequently by loss of ketene from 6. Long-aliphatic-chained alkene 7 undergoes further decomposition to produce the lower molecular weight alkenes ethene, propene, butadiene, and aromatic benzene. Importantly, many of these alkenes and benzene are known carcinogenic constituents within tobacco smoke (22, 23) and elevated amounts of their oxidative metabolites have been found in a study of adolescent e-cigarette users (24). Both pathways produced duroquinone 8, which is an organic oxidant although insufficient data are currently available of its inhalation hazards (25). The high ketene reactivity prevented its NMR detection within the isolated complex volatile mixture. Cumulatively, theoretical DFT calculations, analytical APCI-MS, and experimental results point toward pathway B being the predominate route of pyrolysis under vaping conditions.

Fig. 9. Rationale of experimental and theoretical results from vape pyrolysis reactions of vitamin E acetate 1.

As direct isolation of ketene under low-temperature conditions proved challenging, chemical trapping experiments were next carried out to verify its formation. Benzylamine 9 was chosen as a representative amine nucleophile as the chemical shift of its methylene group (s, 3.85 ppm) appears in a spectral region devoid of peaks in the vape mixture (between 4.6 and 3.8 ppm). Experimentally, the vaped mixture of 1 was passed through a trap containing a solution of benzylamine in CDCl 3 at room temperature. Subsequent NMR analysis clearly showed an additional doublet at 4.36 ppm (not present in the vape mixture alone) which was consistent with an authentic sample of the acetylation benzylamide product 10 (Fig. 10 and SI Appendix, Figs. S15 and S16). Integration of the benzylamide methylene peak, from three independent experiments, with that of the methyl signal for 8 approximated that 30% of ketene produced had reacted with 9. The formation of 10 was confirmed by HPLC and GC-MS experiments (SI Appendix, Figs. S17 and S18).

Fig. 10. Chemical trapping of vaped produced ketene with benzylamine 9 forming benzylamide 10. Portion of the 1H NMR spectra showing vape mixture from 1 isolated in CDCl 3 containing 9 and an authentic sample of benzylamide 10. Black asterisk, methylene peak of 9; red asterisk, methylene peak of 10. See SI Appendix, Figs. S15 and S16 for full spectra.

The current worrying trend of increasing vaping-associated lung injuries is due to complex and multifaceted issues encompassing social, physical, biological, and medical sciences. When viewed from a physical science standpoint, medical complications from vaping a diverse set of substrates is perhaps not unexpected as the pyrolytic chemistry of single pure compounds is complex, so what occurs within ill-defined mixtures is a risky venture into the unknown. The temperatures obtainable within vaping devices places them in the category of a small-scale laboratory pyrolysis apparatus which if not used with precision and care can have unforeseen outcomes. In this article, we have highlighted the chemical basis for concern over vitamin E acetate, just one known component of vape mixtures. Our primary concern initially lay with the ability of the aryl acetate functional group to eliminate as ketene, although other reactive and known carcinogen (alkenes, benzene) are also produced for which the negative long-term effects are well recognized. Thermal activation of aryl acetates above their decomposition temperatures leads to the formation of highly toxic ketene, which if inhaled into the lungs, even in small quantities, can cause severe pulmonary injury. Evidence for ketene formation from vitamin E acetate was obtained from APCI-MS, DFT calculations, and trapping experiments. In closing, it is important to note that it is most likely that other aryl O-acetates would eliminate ketene in a similar manner to the two substrates investigated in this article. Additionally, published data on the pyrolysis of flavor ingredients and additives used in vape products verified the generation of a spectrum of known carcinogens (26). Considering the continuing evolving and large number of natural and synthetic substances used in recreational vaping and the unknown chemistries that may occur under vaping pyrolysis conditions, urgent research into this topic is now required. The most efficient approach would appear to be the development of pyrolysis prediction software, cross-referenced with toxicity data, allowing the rapid identification of individual compounds and compound mixtures that pose the most serious risks.