Hyperpolarised NMR to aid molecular profiling of electronic cigarette aerosols

Signal amplification by reversible exchange (SABRE) hyperpolarisation is used to enhance the NMR signals of nicotine and acrolein in methanol-d4 solutions of electronic cigarette aerosols. Consequently, detection of 74 μM nicotine is possible in just a single scan 1H NMR spectrum. The first example of an aldehyde hyperpolarised using SABRE is demonstrated and we work towards novel real-world applications of SABRE-hyperpolarised NMR for chemical analysis.


S1.1 General Remarks
All NMR measurements were carried out on a 400 MHz Bruker Avance III spectrometer using solutions at room temperature (298 K). 1 H (400 MHz) and 13 C (100.6 MHz) NMR spectra were recorded with an internal deuterium lock. Chemical shifts are quoted as parts per million and referenced to the solvent. 13 C NMR spectra were recorded with broadband proton decoupling. Absolute values of coupling constants (J) are quoted in Hertz and rounded to the nearest 0.5 Hz.
Samples were prepared containing either 2 or 0.8 mg [IrCl(COD)(IMes)] precatalyst and the indicated substrate (nicotine) and/or coligand in 0.5 or 0.6 mL of methanol-d4 in a 5 mm NMR tube that was fitted with a quick pressure valve. The resulting solutions were degassed by three freeze-pump-thaw cycles before the addition of 3-bar pH2. In cases where nicotine, or electronic cigarette aerosol solutions, were added to samples containing SABRE active catalyst the solution was frozen in liquid nitrogen before the valve was removed and the addition made. The solution was then degassed using a vacuum pump to remove air and the solution melted.

S1.2 Preparation of Ecig and Ecig diluent samples
Two samples were prepared. Ecig contained aerosol of electronic cigarette fluid containing 18 mg/mL nicotine mixed with 50:50 VG18 and PG18 NicBase where VG and PG are vegetable glycerine and propylene glycol respectively (Chemnovatic, Lublin, Poland). Ecig diluent consisted only of aerosol of the electronic cigarette carrier fluid 50VG:50PG (Lubrisolve, Somerton, UK). Both liquids were heated with eGO AIO vaporizer (Joyetech Electronics Co., Ltd., Shenzhen, China) for 12 x 10 second pulses and the vapour was led through 2 mL methanol-d4 using a vacuum system. A photograph of the apparatus used to prepare these aerosol solutions is shown in Figure S1.

S1.3 Formation and utilisation of parahydrogen
Hydrogen gas was produced from the electrolysis of water using a desktop hydrogen generator (F-DGSi, Evry, France). This was used directly to make parahydrogen (pH2) using a BPHG 90 parahydrogen generator (Bruker) which passes the hydrogen gas over a spin-exchange catalyst at low temperature. The generator operates at ca 38 K and produces a constant flow of pH2 with ca 92% purity. Parahydrogen (3 bar) was added to NMR tubes containing a quick pressure valve using a home-built system shown in Figure  S2. This set-up contains 3 valves that allow the system to be opened to A) the NMR tube B) parahydrogen and C) a vacuum pump. This system was used to degas the sample (see Section S1.1).

Figure S2: Picture of a) hydrogen generator b) parahydrogen generator and c) home-built device for addition of parahydrogen to NMR tubes in which A, B and C refer to the valves that allow opening of the system to the NMR tube, parahydrogen line or vacuum pump respectively.
Samples were left to activate for a period of several hours (typically around 16 hours overnight). Activation is usually accompanied by a change in colour from orange to pale orange. Catalyst activation is indicated in 1 H NMR spectra by the formation of a peak corresponding to [Ir(H)2(IMes)(L)3]Cl between δ= −21 and −24 ppm. As catalyst activation proceeds, 1 H NMR signals for the COD ligand at δ = 3 and 4 ppm eventually disappear and a signal for cyclooctane at ca δ = 1.6 ppm becomes visible. [2][3][4] When solutions involving catalyst loadings of 5 mM are used, catalyst activation to form [Ir(H)2(IMes)(imidazole)3]Cl generally gives rise to solutions containing a single hydride 1 H NMR peak in the region at δ = −22.34 ppm. 5 We note that under dilute conditions (2 mM catalyst loadings) activation of [Ir(H)2(IMes)(imidazole)3]Cl proceeds less cleanly. In these solutions, other hydride 1 H NMR signals in addition to those of [Ir(H)2(IMes)(imidazole)3]Cl are observed. We attribute this to formation of solvent bound adducts such as [Ir(H)2(IMes)(imidazole)2(ODCD3)]Cl which are likely responsible for a signal sometimes observed at δ = −28.87 ppm or other impurity bound adducts which appear more likely to form at higher solvent:catalyst ratios. These side products appear to reduce SABRE efficiency 6,7 of imidazole and therefore in the experiments detailed here we discard solutions that contain significant amounts of these impurities.

S1.4 SABRE hyperpolarisation measurements
The shake and drop method was employed for recording hyperpolarised NMR spectra. This involves filling NMR tubes with fresh pH2 (3 bar) as described in Section S1.2 before shaking them vigorously for 10 seconds in a 6.5 mT magnetic field. 2 We find that the stray field of our shielded 9.4 T magnet is no larger than 2 mT, therefore we use an electromagnetic coil powered by a Blanko PS-3005 0-30V 0-5A switching power supply to provide the necessary magnetic fields for SABRE polarisation transfer. The current and voltage of the power supply can be altered to achieve a magnetic field inside the coil of ca 6.5 mT. Magnetic fields were measured using a Hirst GM04 Gaussmeter and we estimate that the sample experiences a magnetic field of 6.5 ± 1.0 mT during the manual shaking process. This set up is shown in Figure S3 and is placed as close to the NMR spectrometer as possible to reduce transfer time after the 10 second shaking period. The sample is inserted into the spectrometer as rapidly as possible, this is facilitated by pre-emptively turning off the lift function on the spectrometer. 1 H NMR pulse sequences are modified to include an autosuspend function such that radiofrequency excitation occurs immediately upon sample insertion. 1 H NMR signal enhancements were calculated by dividing the hyperpolarised signal integral intensity by their corresponding integrals in a thermally polarised spectrum. It is essential that both spectra are recorded and processed using the same spectral acquisition parameters. Unless otherwise stated, multiple shake and drop measurements were undertaken and average 1 H NMR signal enhancement values quoted. Signal to noise ratios were calculated using the topspin 'sino' command which takes the largest intensity value of a signal within a given frequency window and divides it by spectral noise within a second frequency window of the same spectrum. The same frequency windows were used throughout this work: for the inequivalent nicotine ortho signals at δ = 8.52 and 8.46 ppm, the maximal signal intensity between δ = 8.53-8.49 and 8.42-8.48 ppm respectively were used with the region between δ = −1 and −5 ppm (which contains no signals) used for the calculation of spectral noise. S2. Analysis of electronic cigarette aerosol solutions using thermally polarised NMR

S2.1 Thermally polarised NMR characterisation of Ecig
Thermally polarised 1D and 2D NMR (COSY, NOESY, HMQC) were used to characterise some of the molecules present within the electronic cigarette aerosol solutions. For these experiments, samples containing electronic cigarette aerosol (100 µL) in methanol-d4 (0.5 mL) were used. NMR characterisation was performed at 9.4 T and 298 K. An example 1 H NMR and 1 H-1 H COSY spectrum is shown in Figure S4 and S5 respectively with NMR characterisation data presented in Table S1.

S2.2 Thermally polarised NMR characterisation of Ecig diluent
Thermally polarised 1D and 2D NMR (COSY, NOESY, HMQC) were used to characterise some of the molecules present within the electronic cigarette aerosol solutions. For these experiments, samples containing Ecig diluent (100 µL) in methanol-d4 (0.5 mL) were used. NMR characterisation was performed at 9.4 T and 298 K. An example 1 H NMR and 1 H-1 H COSY spectrum is shown in Figure  S6 and S7 respectively.  Figure S7: Partial 1 H-1 H COSY spectra recorded at 9.4 T and 298 K of a solution of Ecig diluent aerosol (100 µL) in methanol-d4 (0.5 mL). Those signals marked by the green symbols are consistent with 1,4-pentadien-3-ol or related molecules.

H and 13 C NMR resonances of [Ir(H)2(IMes)(nicotine)3]Cl collected at 9.4 T and 298 K in methanol-d4 with the resonance positions corresponding to those shown in
*Two resonances are observed, these effects have been observed in other works and have been attributed to the inequivalence of the two nicotine ligands which is a result of nicotine chirality. 8,9 Resonance

S3.3 SABRE hyperpolarisation of nicotine
A solution of [Ir(H)2(IMes)(nicotine)3]Cl was shaken vigorously with pH2 (3 bar) for 10 seconds at 6.5 mT before rapid sample insertion into a 9.4 T NMR spectrometer for collection of a single scan 1 H NMR spectrum. These spectra yield enhanced 1 H NMR signals at δ = 8.52, 8.46, 7.45 and 7.88 ppm corresponding to the two inequivalent ortho, meta and para resonances of the free nicotine pyridine ring respectively which are 66 ± 9, 65 ± 12, 107 ± 19 and 87 ± 17 times larger than those recorded under Boltzmann conditions respectively ( Figure S12). None of the 1 H NMR signals of the nicotine pyrrolidine ring were hyperpolarised in these measurements. An enhanced hydride signal for [Ir(H)2(IMes)(nicotine)3]Cl at δ = −22.67 ppm is also observed. Signals for nicotine located trans to hydrides within [Ir(H)2(IMes)(nicotine)3]Cl are also enhanced with 1 H NMR signal gains of 65 ± 12, 78 ± 17, 41 ± 8 and 58 ± 13 -fold for the two ortho, meta and para sites respectively ( Figure S12).

S4.1 SABRE Hyperpolarised NMR of Ecig
A solution containing [IrCl(COD)(IMes)] (5 mM) (where COD is cis,cis-1,5-cyclooctadiene and IMes is 1,3-bis(2,4,6-trimethylphenyl)imidazol-2-ylidene) and imidazole (15 equiv.) in methanol-d4 (0.5 mL) was activated with H2 (3 bar) for a few hours at room temperature. At this point 50 µL of the electronic cigarette aerosol solution was added before pH2 shaking was performed (3 bar for 10 seconds at 6.5 mT). Later, a second 50 µL addition of the electronic cigarette aerosol solution was performed. After these experiments were performed, many scan reference 1 H NMR spectra were collected and compared to analogous measurements recorded on a sample containing the same electronic cigarette aerosol solution (100 µL) in methanol-d4 (0.5 mL). A comparison between these spectra is shown in Figure S14 and shows that acrolein signals are no longer present in the solution containing the SABRE catalyst. More detailed 2D NMR characterisation of the electronic cigarette aerosol sample is presented in Section S2.

S4.2 SABRE Hyperpolarised NMR of Ecig diluent
A solution containing [IrCl(COD)(IMes)] (5 mM) (where COD is cis,cis-1,5-cyclooctadiene and IMes is 1,3-bis(2,4,6-trimethylphenyl)imidazol-2-ylidene) and imidazole (15 equiv.) in methanol-d4 (0.5 mL) was activated with H2 (3 bar) for a few hours at room temperature. At this point 50 µL of the electronic cigarette aerosol solution was added before pH2 shaking was performed (3 bar for 10 seconds at 6.5 mT). Later, a second 50 µL addition of the electronic cigarette aerosol solution was performed. SABREhyperpolarised spectra are shown in Figure S15. A time course for the hyperpolarised acrolein signals is shown in Figure S16. Estimates of hyperpolarised T1's of acrolein could be determined by recording a series of single scan SABRE-hyperpolarised 1 H NMR spectra with 10 degree flip angles. The decay of hyperpolarised signal intensity over time is shown in Figure S17 and occurs due to radiofrequency excitation and nuclear spin relaxation. This can be accounted for using the following equations to extract a T1.

Figure S15: Partial single scan 1 H NMR spectra recorded at 9.4 T and 298 K of a) [IrCl(COD)(IMes)] (5 mM) and imidazole (15 equiv.) in 0.5 mL methanol-d4 shaken with 3 bar pH2 for 10 seconds at 6.5 mT. b) SABRE HP spectra of the solution used in a) ca 5 minutes after addition of 50 µL Ecig diluent . c) Thermally polarised 1 H NMR spectrum of the solution used in b) ca 10 minutes after the addition of Ecig diluent to the solution used in a). Note that signals denoted 'Im' and 'C' refer to imidazole and the IMes ligand of the SABRE catalyst respectively and that c) is expanded vertically by a factor of 8 relative to a) and b). Those signals marked by the green symbols are consistent with 1,4-pentadien-3-ol or related molecules.
In this model the hyperpolarized signal of species A, ( ) , detected by the 10° pulse at time t is calculated according to Equation 1, where ( ) is the magnetization of species A at time t and θ is the flip angle. The magnetization of species A remaining after the pulse is given by Equation 2. The magnetization of A changes during the time interval between successive pulses according to nuclear spin relaxation, as described in Equation 3. Values of ( ) were calculated from the model and compared to values determined from experiments. T1 was found by minimising the least squared summed differences between experimental and calculated values. Similar approaches have been used elsewhere. 12 In this example the model gives only an estimate for T1 as it does not account for decay of hyperpolarised signals due to chemical reaction (such as binding to the metal centre, hydrogenation or other decomposition). This should be a valid assumption as the reaction rate is expected to be significantly slower than the relaxation rate. (i.e. reaction under these conditions occurred over a ca 40 minute time window, see Figure S16), therefore within this 40 second observation window the dominant processes leading to signal decay that must be accounted for are radiofrequency excitation and relaxation and chemical reaction has therefore been omitted.