Detection, discrimination and quantification of amphetamine, cathinone and nor-ephedrine regioisomers using benchtop 1 H and 19 F nuclear magnetic resonance spectroscopy.

Amphetamine and cathinone derivatives are abused recreationally due to the sense of euphoria they provide to the user. Methodologies for the rapid detection of the drug derivative present in a seized sample, or an indication of the drug class, are beneficial to law enforcement and healthcare providers. Identifying the drug class is prudent because derivatisation of these drugs, to produce regioisomers, for example, occurs frequently to circumvent global and local drug laws. Thus, newly encountered derivatives might not be present in a spectral library. Employment of benchtop nuclear magnetic resonance (NMR) could be used to provide rapid analysis of seized samples as well as identifying the class of drug present. Discrimination of individual amphetamine-, methcathinone-, N-ethylcathinone and nor-ephedrine-derived fluorinated and methylated regioisomers is achieved herein using qualitative automated 1 H NMR analysis and compared to gas chromatography-mass spectrometry (GC-MS) data. Two seized drug samples, SS1 and SS2, were identified to contain 4-fluoroamphetamine by 1 H NMR (match score median = 0.9933) and GC-MS (RRt = 5.42-5.43 min). The amount of 4-fluoroamphetamine present was 42.8%-43.4% w/w and 48.7%-49.2% w/w for SS1 and SS2, respectively, from quantitative 19 F NMR analysis, which is in agreement with the amount determined by GC-MS (39.9%-41.4% w/w and 49.0%-49.3% w/w). The total time for the qualitative 1 H NMR and quantitative 19 F NMR analysis is ~10 min. This contrasts to ~40 min for the GC-MS method. The NMR method also benefits from minimal sample preparation. Thus, benchtop NMR affords rapid, and discriminatory, analysis of the drug present in a seized sample.

Rapid SABRE Catalyst Scavenging Using Functionalized Silicas.

In recent years the NMR hyperpolarisation method signal amplification by reversible exchange (SABRE) has been applied to multiple substrates of potential interest for in vivo investigation. Unfortunately, SABRE commonly requires an iridium-containing catalyst that is unsuitable for biomedical applications. This report utilizes inductively coupled plasma-optical emission spectroscopy (ICP-OES) to investigate the potential use of metal scavengers to remove the iridium catalytic species from the solution. The most sensitive iridium emission line at 224.268 nm was used in the analysis. We report the effects of varying functionality, chain length, and scavenger support identity on iridium scavenging efficiency. The impact of varying the quantity of scavenger utilized is reported for the three scavengers with the highest iridium removed from initial investigations: 3-aminopropyl (S1), 3-(imidazole-1-yl)propyl (S4), and 2-(2-pyridyl) (S5) functionalized silica gels. Exposure of an activated SABRE sample (1.6 mg mL-1 of iridium catalyst) to 10 mg of the most promising scavenger (S5) resulted in <1 ppm of iridium being detectable by ICP-OES after 2 min of exposure. We propose that combining the approach described herein with other recently reported approaches, such as catalyst separated-SABRE (CASH-SABRE), would enable the rapid preparation of a biocompatible SABRE hyperpolarized bolus.

Hyperpolarisation of Mirfentanil by SABRE in the Presence of Heroin.

Mirfentanil, a fentanyl derivative that is a µ-opioid partial agonist, is hyperpolarised via Signal Amplification By Reversible Exchange (SABRE), a para-hydrogen-based technique. [Ir(IMes)(COD)Cl] (IMes=1,3-bis(2,4,6-trimethylphenyl)imidazole-2-ylidene, COD=cyclooctadiene) was employed as the polarisation transfer catalyst. Following polarisation transfer at 6.5 mT, the pyrazine-protons were enhanced by 78-fold (polarisation, P=0.04 %). The complex [Ir(IMes)(H)2 (mirfentanil)2 (MeOH)]+ is proposed to form based on the observation of two hydrides at δ -22.9 (trans to mirfentanil) and -24.7 (trans to methanol). In a mixture of mirfentanil and heroin, the former could be detected using SABRE at concentrations less than 1 % w/w. At the lowest concentration analyzed, the amount of mirfentanil present was 0.18 mg (812 µM) and produced a signal enhancement of -867-fold (P=0.42 %). following polarisation transfer at 6.5 mT.

Delivering strong 1H nuclear hyperpolarization levels and long magnetic lifetimes through signal amplification by reversible exchange.

Hyperpolarization turns typically weak NMR and MRI responses into strong signals so that ordinarily impractical measurements become possible. The potential to revolutionize analytical NMR and clinical diagnosis through this approach reflect this area's most compelling outcomes. Methods to optimize the low-cost parahydrogen-based approach signal amplification by reversible exchange with studies on a series of biologically relevant nicotinamides and methyl nicotinates are detailed. These procedures involve specific 2H labeling in both the agent and catalyst and achieve polarization lifetimes of ca 2 min with 50% polarization in the case of methyl-4,6-d2 -nicotinate. Because a 1.5-T hospital scanner has an effective 1H polarization level of just 0.0005% this strategy should result in compressed detection times for chemically discerning measurements that probe disease. To demonstrate this technique's generality, we exemplify further studies on a range of pyridazine, pyrimidine, pyrazine, and isonicotinamide analogs that feature as building blocks in biochemistry and many disease-treating drugs.

Benchtop NMR analysis of piperazine-based drugs hyperpolarised by SABRE.

Piperazine-based drugs, such as N-benzylpiperazine (BZP), became attractive in the 2000s due to possessing effects similar to amphetamines. Herein, BZP, in addition to its pyridyl analogues, 2-, 3-, and 4-pyridylmethylpiperidine (2-PMP, 3-PMP, and 4-PMP respectively) was subjected to the hyperpolarisation technique Signal Amplification By Reversible Exchange (SABRE) in order to demonstrate the use of this technique to detect these piperazine-based drugs. Although BZP was not hyperpolarised via SABRE, 2-PMP, 3-PMP, and 4-PMP were, with the ortho- and meta-pyridyl protons of 4-PMP showing the largest enhancement of 313-fold and 267-fold, respectively, in a 1.4-T detection field, following polarisation transfer at Earth's magnetic field. In addition to the freebase, 4-PMP.3HCl was also appraised by SABRE and was found not to polarise, however, the addition of increasing equivalents of triethylamine (TEA) produced the freebase, with a maximum enhancement observed upon the addition of 3 equivalents of TEA. Further addition of TEA led to a reduction in the observed enhancement. SABRE was also employed to polarise 4-PMP.3HCl (~20% w/w) in a simulated tablet to demonstrate the forensic application of the technique (138-fold enhancement for the ortho-pyridyl protons). The amount of 4-PMP.3HCl present in the simulated tablet was quantified via NMR using D2 O as a solvent and compared well to complimentary gas chromatography-mass spectrometry data. Exchanging D2 O for CD3 OD as the solvent utilised for analysis resulted in a significantly lower amount of 4-PMP.3HCl being determined, thus highlighting safeguarding issues linked to drug abuse in relation to determining the amount of active pharmaceutical ingredient present.

Using 2 H labelling to improve the NMR detectability of pyridine and its derivatives by SABRE.

By introducing a range of 2 H labels into pyridine and the para-substituted agents, methyl isonicotinate and isonicotinamide, we significantly improve their NMR detectability in conjunction with the signal amplification by reversible exchange process. We describe how the rates of T1 relaxation for the remaining 1 H nuclei are increased and show how this leads to a concomitant increase in the level of 1 H and 13 C hyperpolarization that can ultimately be detected.

Synthesis and hyperpolarisation of eNOS substrates for quantification of NO production by 1H NMR spectroscopy.

Hyperpolarization enhances the intensity of the NMR signals of a molecule, whose in vivo metabolic fate can be monitored by MRI with higher sensitivity. SABRE is a hyperpolarization technique that could potentially be used to image nitric oxide (NO) production in vivo. This would be very important, because NO dysregulation is involved in several pathologies, including cardiovascular ones. The nitric oxide synthase (NOS) pathway leads to NO production via conversion of l-arginine into l-citrulline. NO is a free radical gas with a short half-life in vivo (≈5s), therefore direct NO quantification is challenging. An indirect method - based on quantifying conversion of an l-Arg- to l-Cit-derivative by 1H NMR spectroscopy - is herein proposed. A small library of pyridyl containing l-Arg derivatives was designed and synthesised. In vitro tests showed that compounds 4a-j and 11a-c were better or equivalent substrates for the eNOS enzyme (NO2- production=19-46µM) than native l-Arg (NO2- production=25µM). Enzymatic conversion of l-Arg to l-Cit derivatives could be monitored by 1H NMR. The maximum hyperpolarization achieved by SABRE reached 870-fold NMR signal enhancement, which opens up exciting future perspectives of using these molecules as hyperpolarized MRI tracers in vivo.

Aspartate-Based CXCR4 Chemokine Receptor Binding of Cross-Bridged Tetraazamacrocyclic Copper(II) and Zinc(II) Complexes.

The CXCR4 chemokine receptor is implicated in a number of diseases including HIV infection and cancer development and metastasis. Previous studies have demonstrated that configurationally restricted bis-tetraazamacrocyclic metal complexes are high-affinity CXCR4 antagonists. Here, we present the synthesis of Cu(2+) and Zn(2+) acetate complexes of six cross-bridged tetraazamacrocycles to mimic their coordination interaction with the aspartate side chains known to bind them to CXCR4. X-ray crystal structures for three new Cu(2+) acetate complexes and two new Zn(2+) acetate complexes demonstrate metal-ion-dependent differences in the mode of binding the acetate ligand concomitantly with the requisite cis-V-configured cross-bridged tetraazamacrocyle. Concurrent density functional theory molecular modelling studies produced an energetic rationale for the unexpected [Zn(OAc)(H2 O)](+) coordination motif present in all of the Zn(2+) cross-bridged tetraazamacrocycle crystal structures, which differs from the chelating acetate [Zn(OAc)](+) structures of known unbridged and side-bridged tetraazamacrocyclic Zn(2+) -containing CXCR4 antagonists.

Developments and advances concerning the hyperpolarisation technique SABRE.

To overcome the inherent sensitivity issue in NMR and MRI, hyperpolarisation techniques are used. Signal Amplification By Reversible Exchange (SABRE) is a hyperpolarisation technique that utilises parahydrogen, a molecule that possesses a nuclear singlet state, as the source of polarisation. A metal complex is required to break the singlet order of parahydrogen and, by doing so, facilitates polarisation transfer to analyte molecules ligated to the same complex through the J-coupled network that exists. The increased signal intensities that the analyte molecules possess as a result of this process have led to investigations whereby their potential as MRI contrast agents has been probed and to understand the fundamental processes underpinning the polarisation transfer mechanism. As well as discussing literature relevant to both of these areas, the chemical structure of the complex, the physical constraints of the polarisation transfer process and the successes of implementing SABRE at low and high magnetic fields are discussed.

Toward biocompatible nuclear hyperpolarization using signal amplification by reversible exchange: quantitative in situ spectroscopy and high-field imaging.

Signal amplification by reversible exchange (SABRE) of a substrate and parahydrogen at a catalytic center promises to overcome the inherent insensitivity of magnetic resonance. In order to apply the new approach to biomedical applications, there is a need to develop experimental equipment, in situ quantification methods, and a biocompatible solvent. We present results detailing a low-field SABRE polarizer which provides well-controlled experimental conditions, defined spins manipulations, and which allows in situ detection of thermally polarized and hyperpolarized samples. We introduce a method for absolute quantification of hyperpolarization yield in situ by means of a thermally polarized reference. A maximum signal-to-noise ratio of ∼10(3) for 148 µmol of substance, a signal enhancement of 10(6) with respect to polarization transfer field of SABRE, or an absolute (1)H-polarization level of ≈10(-2) is achieved. In an important step toward biomedical application, we demonstrate (1)H in situ NMR as well as (1)H and (13)C high-field MRI using hyperpolarized pyridine (d3) and (13)C nicotinamide in pure and 11% ethanol in aqueous solution. Further increase of hyperpolarization yield, implications of in situ detection, and in vivo application are discussed.

Probing signal amplification by reversible exchange using an NMR flow system.

Hyperpolarization methods are used in NMR to overcome its inherent sensitivity problem. Herein, the biologically relevant target nicotinamide is polarized by the hyperpolarization technique signal amplification by reversible exchange. We illustrate how the polarization transfer field, and the concentrations of parahydrogen, the polarization-transfer-catalyst and substrate can be used to maximize signal amplification by reversible exchange effectiveness by reference to the first-order spin system of this target. The catalyst is shown to be crucial in this process, first by facilitating the transfer of hyperpolarization from parahydrogen to nicotinamide and then by depleting the resulting polarized states through further interaction. The 15 longitudinal one, two, three and four spin order terms produced are rigorously identified and quantified using an automated flow apparatus in conjunction with NMR pulse sequences based on the only parahydrogen spectroscopy protocol. The rates of build-up of these terms were shown to follow the order four~three > two > single spin; this order parallels their rates of relaxation. The result of these competing effects is that the less-efficiently formed single-spin order terms dominate at the point of measurement with the two-spin terms having amplitudes that are an order of magnitude lower. We also complete further measurements to demonstrate that (13)C NMR spectra can be readily collected where the long-lived quaternary (13)C signals appear with significant intensity. These are improved upon by using INEPT. In summary, we dissect the complexity of this method, highlighting its benefits to the NMR community and its applicability for high-sensitivity magnetic resonance imaging detection in the future.

Application of parahydrogen induced polarization techniques in NMR spectroscopy and imaging.

Magnetic resonance provides a versatile platform that allows scientists to examine many different types of phenomena. However, the sensitivity of both NMR spectroscopy and MRI is low because the detected signal strength depends on the population difference that exists between the probed nuclear spin states in a magnetic field. This population difference increases with the strength of the interacting magnetic field and decreases with measurement temperature. In contrast, hyperpolarization methods that chemically introduce parahydrogen (a spin isomer of hydrogen with antiparallel spins that form a singlet) based on the traditional parahydrogen induced polarization (PHIP) approach tackle this sensitivity problem with dramatic results. In recent years, the potential of this method for MRI has been recognized, and its impact on medical diagnosis is starting to be realized. In this Account, we describe the use of parahydrogen to hyperpolarize a suitable substrate. This process normally involves the introduction of a molecule of parahydrogen into a target to create large population differences between nuclear spin states. The reaction of parahydrogen breaks the original magnetic symmetry and overcomes the selection rules that prevent both NMR observation and parahydrogen/orthohydrogen interconversion, yielding access to the normally invisible hyperpolarization associated with parahydrogen. Therefore the NMR or MRI measurement delivers a marked increase in the detected signal strength over the normal Boltzmann-population derived result. Consequently, measurements can be made which would otherwise be impossible. This approach was pioneered by Weitekamp, Bargon, and Eisenberg, in the late 1980s. Since 1993, we have used this technique in York to study reaction mechanisms and to characterize normally invisible inorganic species. We also describe signal amplification by reversible exchange (SABRE), an alternative route to sensitize molecules without directly incorporating a molecule of parahydrogen. This approach widens the applicability of PHIP methods and the range of materials that can be hyperpolarized. In this Account we describe our parahydrogen studies in York over the last 20 years and place them in a wider context. We describe the characterization of organometallic reaction intermediates including those involved in catalytic reactions, either with or without hydride ligands. The collection of spectroscopic and kinetic data with rapid inverse detection methods has proved to be particularly informative. We can see enhanced signals for the organic products of catalytic reactions that are linked directly to the catalytic intermediates that form them. This method can therefore prove unequivocally that a specific metal complex is involved in a catalytic cycle, thus pinpointing the true route to catalysis. Studies where a pure nuclear spin state is detected show that it is possible to detect all of the analyte molecules present in a sample using NMR. In addition, we describe methods that achieve the selective detection of these enhanced signals, when set against a strong NMR background such as that of water.

Improving NMR and MRI sensitivity with parahydrogen.

Parahydrogen induced polarisation (PHIP) has wide utility in NMR and MRI as it can increase the sensitivity of both techniques. The transfer of spin order from parahydrogen to nuclei in the analyte leads to an increased magnetic response following interrogation by RF pulses. This spin transfer is catalysed by a homogeneous or heterogeneous catalyst. The increased magnetic response not only reduces the number of transients required to obtain the spectrum or image, but can also illuminate previously undetectable species present in solution. From its theoretical prediction to its experimental validation, PHIP has been applied in a range of different areas such as the structural analysis of complexes, understanding reaction mechanisms involving hydrogen and for the production of contrast agents for use in MRI. PHIP can also be readily combined with other techniques such as photochemistry which widens its field of applicability. In this review, we detail the properties of parahydrogen and the methods for its preparation and utilisation in homogeneous and heterogeneous based hydrogenation and non-hydrogenative reactions. Specific examples are explained for the application of PHIP in photochemical and hydroformylation reactions. Pulse sequences designed to be compatible with PHIP are described to exemplify how the increase in sensitivity can be increased even further by the interrogation of the magnetic states optimally. Finally, a section on the use of PHIP in the production of contrast agents suitable for MRI, and the monitoring of hydrogenation reactions using imaging techniques is discussed.

Iridium(III) hydrido N-heterocyclic carbene-phosphine complexes as catalysts in magnetization transfer reactions.

The hyperpolarization (HP) method signal amplification by reversible exchange (SABRE) uses para-hydrogen to sensitize substrate detection by NMR. The catalyst systems [Ir(H)2(IMes)(MeCN)2(R)]BF4 and [Ir(H)2(IMes)(py)2(R)]BF4 [py = pyridine; R = PCy3 or PPh3; IMes = 1,3-bis(2,4,6-trimethylphenyl)imidazol-2-ylidene], which contain both an electron-donating N-heterocyclic carbene and a phosphine, are used here to catalyze SABRE. They react with acetonitrile and pyridine to produce [Ir(H)2(NCMe)(py)(IMes)(PPh3)]BF4 and [Ir(H)2(NCMe)(py)(IMes)(PCy3)]BF4, complexes that undergo ligand exchange on a time scale commensurate with observation of the SABRE effect, which is illustrated here by the observation of both pyridine and acetonitrile HP. In this study, the required symmetry breaking that underpins SABRE is provided for by the use of chemical inequivalence rather than the previously reported magnetic inequivalence. As a consequence, we show that the ligand sphere of the polarization transfer catalyst itself becomes hyperpolarized and hence that the high-sensitivity detection of a number of reaction intermediates is possible. These species include [Ir(H)2(NCMe)(py)(IMes)(PPh3)]BF4, [Ir(H)2(MeOH)(py)(IMes)(PPh3)]BF4, and [Ir(H)2(NCMe)(py)2(PPh3)]BF4. Studies are also described that employ the deuterium-labeled substrates CD3CN and C5D5N, and the labeled ligands P(C6D5)3 and IMes-d22, to demonstrate that dramatically improved levels of HP can be achieved as a consequence of reducing proton dilution and hence polarization wastage. By a combination of these studies with experiments in which the magnetic field experienced by the sample at the point of polarization transfer is varied, confirmation of the resonance assignments is achieved. Furthermore, when [Ir(H)2(pyridine-h5)(pyridine-d5)(IMes)(PPh3)]BF4 is examined, its hydride ligand signals are shown to become visible through para-hydrogen-induced polarization rather than SABRE.

Utilization of SABRE-derived hyperpolarization to detect low-concentration analytes via 1D and 2D NMR methods.

The characterization of materials by the inherently insensitive method of NMR spectroscopy plays a vital role in chemistry. Increasingly, hyperpolarization is being used to address the sensitivity limitation. Here, by reference to quinoline, we illustrate that the SABRE hyperpolarization technique, which uses para-hydrogen as the source of polarization, enables the rapid completion of a range of NMR measurements. These include the collection of (13)C, (13)C{(1)H}, and NOE data in addition to more complex 2D COSY, ultrafast 2D COSY and 2D HMBC spectra. The observations are made possible by the use of a flow probe and external sample preparation cell to re-hyperpolarize the substrate between transients, allowing repeat measurements to be made within seconds. The potential benefit of the combination of SABRE and 2D NMR methods for rapid characterization of low-concentration analytes is therefore established.

Guilty by dissociation: Part B: evaluation of Supercritical Fluid Chromatography (SFC-UV) for the analysis of regioisomeric diphenidine-derived Novel Psychoactive Substances (NPS).

Supercritical Fluid Chromatography (SFC-UV) employing a carbon dioxide (CO2) and 10 mM ammonium acetate in MeOH-water (95:5 v/v) gradient provides a rapid analysis (tG <10 min) of 31 novel, regioisomeric diphenidine-derived psychoactive substances, on a range of stationary phases of differing polarity. Medium to large selectivity differences between regioisomers, were observed on the acidic, neutral and basic SFC phases. For individual substituted ortho-, meta- and para-isomers, the same elution order was observed irrespective of the nature of the stationary phase. The acidic silica stationary phases yielded longer retention of the diphenidines via electrostatic attraction, whereas the basic phases resulted in shorter retention via electrostatic repulsion. SFC effected baseline separation of seven of the eight substituted groups of ortho-, meta- and para-diphenidines evaluated on a range of stationary phases. A simple silica phase achieved baseline separation of six of the regioisomeric substituted diphenidines. As the size of the halo-substituent increased, the resolution between ortho-/meta-isomers decreased, resulting in co-elution of the ortho- and meta-bromodiphenidines. Fluphenidines and chlorodiphenidines generated an elution order of meta- < ortho- < para- whereas an elution order switch was observed for the iodophenidines. This contrasted with RP-UHPLC where the elution order for the fluphenidines and iodophenidines was para- < ortho- < meta- and para- < meta- < ortho- respectively. An orthogonal elution order of diphenidines was demonstrated between the RP-UHPLC and SFC stationary phases due to the polarity differences between the separation modes. In general, hydrophilic compounds, which were poorly retained on a C18 reverse phase column, were well retained on SFC columns.

Guilty by dissociation: Part A: Development of a rapid Ultra-High Performance Liquid Chromatography (UHPLC)-MS/MS methodology for the analysis of regioisomeric diphenidine-derived Novel Psychoactive Substances (NPS).

This study describes the first reported development of a rapid, generic gradient Ultra-High Performance Liquid Chromatography (UHPLC) methodology with targeted triple quadrupole MS/MS using electrospray positive ionisation to detect and unambiguously confirm the identity of 33 substituted 1, 2-diarylethamine (or diphenidine) derivatives in solid drug samples. The in-house synthesised library included a range of derivatives possessing either electron donating/withdrawing substituents, commonly included in combinatorial libraries, of varying size and lipophilicity on the phenyl ring. These test probes were used to investigate if their order of elution and that of their regioisomers were dependent on the position and type of the substituent on the phenyl ring. In addition, investigations into the retention mechanism of the diphenidines under reverse-phase UHPLC conditions were undertaken. Common adulterants found within seized bulk samples were assessed to prove that the methodology was specific, and the developed UHPLC-MS/MS (tG = 10 min) protocol was applied to confirm the identity of the psychoactive components within four seized bulk samples provided by law enforcement.

Comparative study of the analysis of seized samples by GC-MS, 1H NMR and FT-IR spectroscopy within a Night-Time Economy (NTE) setting.

Rapid analysis of surrendered or seized drug samples provides important intelligence for health (e.g. treatment or harm reduction), and custodial services. Herein, three in-situ techniques, GC-MS, 1H NMR and FT-IR spectroscopy, with searchable libraries, are used to analyse 318 samples qualitatively, using technique specific library-based searches, obtained over the period 24th - 29th August 2019. 259 samples were identified as consisting of a single component, of which cocaine was the most prevalent (n = 158). Median match scores for all three techniques were ≥ 0.84 and showed agreement except for metformin (n = 1), oxandrolone (identified as vitamin K by IR (n = 4)), diazepam (identified as zolpidem by FT-IR (n = 2)) and 2-Br-4,5-DMPEA (n = 1), a structural isomer of 2C-B identified as a polymer of cellulose (cardboard) by FT-IR. 51 samples were found to consist of two or more components, of which 49 were adulterated cocaine samples (45 binary and 4 tertiary samples). GC-MS identified all components present in the 49 adulterated cocaine samples, whereas IR identified only cocaine in 88 % of cases (adulterant only = 12 %). The breakdown for 1H NMR spectroscopy was all components identified (51 %), cocaine only (33 %), adulterant only (10 %), cocaine and one adulterant (tertiary mixtures only, 6 %).

Iridium N-heterocyclic carbene complexes as efficient catalysts for magnetization transfer from para-hydrogen.

While the characterization of materials by NMR is hugely important in the physical and biological sciences, it also plays a vital role in medical imaging. This success is all the more impressive because of the inherently low sensitivity of the method. We establish here that [Ir(H)(2)(IMes)(py)(3)]Cl undergoes both pyridine (py) loss as well as the reductive elimination of H(2). These reversible processes bring para-H(2) and py into contact in a magnetically coupled environment, delivering an 8100-fold increase in (1)H NMR signal strength relative to non-hyperpolarized py at 3 T. An apparatus that facilitates signal averaging has been built to demonstrate that the efficiency of this process is controlled by the strength of the magnetic field experienced by the complex during the magnetization transfer step. Thermodynamic and kinetic data combined with DFT calculations reveal the involvement of [Ir(H)(2)(η(2)-H(2))(IMes)(py)(2)](+), an unlikely yet key intermediate in the reaction. Deuterium labeling yields an additional 60% improvement in signal, an observation that offers insight into strategies for optimizing this approach.