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Application Note as an Adjunct to Spectrometry in Drug Development

Ian N. Acworth, David Thomas, Paul Gamache

Thermo Fisher Scientific, Chelmsford, MA 1016

Key Words Drug bioactivation, Stability and degradation, Electrochemical detection, Enhanced , Drug metabolites, Reactive intermediate

Goal To show how on-line HPLC-electrochemical oxidation- can be harnessed to accelerate several key steps in drug development.

Introduction Oxidation plays a number of critical roles in biology and drug metabolism. In addition to being the major process for generating energy in a living cell, it is the major mechanism by which drugs are metabolized. Oxidation is a contributor to drug bioactivation and idiosyncratic Experimental toxicity, and is also involved in drug stability and Applications were developed using gradient analytical or degradation. Electrochemistry (EC) when used with HPLC semi-preparative HPLC with pre- or post-column has long been recognized as a sensitive and selective electrochemical oxidation followed by mass spectrometric detection technique. Indeed more than 90% of detection. Drug stability studies used flow injection pharmaceuticals in the marketplace can be oxidized and analysis with coulometric array detection. detected using this technique. However, the inherent electrochemical nature of many drugs goes beyond simple Samples were dissolved in initial mobile phase to analysis. This commonality of oxidation makes EC a of 20 µg/mL. particularly well suited to studying the oxidative processes in pharmaceuticals, both in the formulation and by the organism. Presented here are techniques that combine EC The experimental setup is shown in Figure 1. The HPLC and MS in a number of formats that can be used: a) to conditions were as follows: enhance ionization, thereby extending the range of Column: C18 (3 µm, 4.6 × 50 mm) compounds measured by LC-MS; b) for the micro-synthesis and identification ofdrug metabolites; c) for the detection of Gradient: 4 min gradient 1% to 80% with constant supporting of 20 mM ammonium acetate (pH 7) reactive intermediates and their conjugates; d) for identifi- or 50 mM and 10 mM ammonium formate cation of potentially problematic molecular “soft spots”; (pH 3.9), 2 mL/min and e) to rapidly study drug stability and predict which antioxidant(s) should be included in formulations. Detector: Thermo Scientific Dionex Coulochem III Electrochemical Detector (with model 5021A Conditioning Cell or model 5125 Synthesis Cell) or Thermo Scientific Dionex CoulArray Multi-Channel (8) Electrochemical Detector 2 Mass Spectrometry Data from EC-assisted ionization of 11 neutral phenols A single quadrupole mass was used. The MS compiled in Table 1 show that all four compounds that conditions were as follows: exhibit a weak ESI- or atmospheric pressure chemical (ESI): Positive ionization (APCI)-MS response at pH 7, and are EC-active, gave a large MS response after upstream EC oxidation. Scan range: 80 to 500 m/z Similarly, all nine compounds that exhibit a weak ESI- or Fragmentor: 70 V APCI-MS response at pH 3.9, and are EC-active, gave a Gain: 1.0 large MS response after the upstream EC oxidation. Threshold: 150 EC-assisted ionization was only attempted when there was no MS response. Step: 0.25 Drying : 12 L/min On-line Micro-synthesis and Identification of Drug Metabolites Nebulizer: 35 psig Placing the coulometric oxidation cell before the HPLC Drying gas : 350 °C column allowed for detailed characterization of the Capillary : 3500 V EC-generated products. Figure 3 shows a total chromatogram obtained for tamoxifen with precolumn Data Analysis EC oxidation at 1000 mV vs. Pd reference . CoulArray™ Data Station software version 3.10 was Several peaks corresponding to oxidation products are utilized for the acquisition, processing and reporting evident. This example illustrates on-line generation and of analytical data. analysis of EC products using LC-MS conditions that are Results and Discussion typical of metabolic studies, e.g., in vitro microsomal analysis. Serial LC-EC-MS can thus be used with neat Enhanced Ionization parent compound for preliminary optimization The experimental setup shown in Figure 1 allows for of LC and MS/MS conditions and subsequent metabolite chromatographic separation followed by a flow split. One analysis in biological samples. By using identical branch of the flow undergoes with a model conditions, EC data may then be used as input to 5021A conditioning cell followed by MS detection, while automated metabolite identification software to aid in a parallel branch passes through a CoulArray 8-channel finding metabolites present in more complex biological cell pack (sequentially higher potential per channel). matrices.1 Figure 2 shows that upstream oxidation of HPLC-separated Furthermore, when the data from an EC-generated , phenol, o-toluidine and BHA (500 mV, Figure 2B) product correspond to that of a biological metabolite, the led to significantly increased MS response when compared EC technique may then be viewed as a selective and rapid to that obtained with the EC cell off (Figure 2A). synthetic route to small quantities of this metabolite. In Figure 2C shows parallel EC-array detection of the this example, a model 5021A conditioning cell was used compounds. In similar experiments, EC reactions were to produce estimated nanogram quantities of metabolites. observed according to the following general rank order Higher-capacity model 5125 synthesis cells provide the (by relative ease of oxidation) o, p-quinol and o, p-amino- ability to produce larger quantities for more detailed phenol > tertiary amine > m-quinol ≈ phenol ≈ arylamine structural elucidation studies. Figure 4 shows that high > secondary amine ≈ thiol > thioether. No EC oxidation efficiency is maintained for oxidation of 2 µg quantities of was observed for primary aliphatic amines and alcohols amitriptyline (AMI) over a range of flow rates, thus and oxidation of secondary and tertiary amines was less facilitating the production of quantities that may be facile at pH 3.9, than at pH 7. These differences in analyzed by nuclear magnetic resonance (NMR). oxidation potential provide a means of increased resolution Oxidation efficiency was calculated by subtracting the and selectivity when using parallel EC-array detection. normalized AMI UV response obtained with the cell on Furthermore, differences in potential may also from that obtained with the cell off (UV response with cell provide a means of distinguishing isobaric species off = 100%). observed by MS based on the use of upstream EC oxidation. This may be particularly useful for drug Detection and Trapping of Reactive Intermediates biotransformation studies to distinguish, for example, Many studies suggest that redox metabolism of a wide aromatic and aliphatic hydroxylation – oxidation of the range of chemical to formation of reactive latter being much less favorable. electrophiles, which participate in a diverse array of toxic processes that typically involve covalent binding or other modifications to small and large . The propensity of compounds to undergo redox-based metabolic activation is therefore a major consideration in pharmaceutical development. Several reports have shown LC-EC-MS useful in the study of reactive intermediate metabolites.2,3,4 Figure 5 provides an example of precolumn EC oxidation 3 using the widely studied compound, acetaminophen (APAP). Oxidative metabolic activation of APAP to form N-acetyl p-benzoquinoneimine (NAPQI) is widely regarded as an essential component of its hepatotoxic effects in humans. MS data indicate that EC oxidation of APAP in the presence of GSH resulted in two separate peaks corresponding to monoglutathionyl conjugates, and one peak indicative of a diglutathionyl conjugate. In this example, 10 µL of a 20 µg/mL acetaminophen/1 mM GSH mixture was injected while the model 5021A cell was held at 500 mV vs. Pd. Figure 6 shows the precolumn oxidation of phenacetin by a model 5021A cell set at 500 mV vs. Pd. The HPLC detector was an 8-channel CoulArray detector with model 6210 EC cell potentials of 0 to 840 mV vs. Pd in 120 mV increments. The peak eluting at 3.8 min exhibits the characteristic voltammetric profile (i.e., reduction followed by oxidation) of a quinone species. EC Array and MS data (not shown) identify this peak as NAPQI, Figure 1. Instrument configuration the expected reactive intermediate. NAPQI was not evident in a microsomal incubate of phenacetin analyzed using the same conditions (not shown), possibly because nonspecific binding of this reactive species occurred in the A) MS EC cell off, Extracted Ion Chromatograms M+H biological preparation. Rapid Identification of Molecular “Soft Spots” The instrumental LC-EC-MS technique provides a rapid means of obtaining information on the relative ease and likely chemical sites of oxidation, as well as the nature of B) Extracted Ion Chromatograms - Serial EC-MS (500 mV) products obtained from these on-line reactions. These data can serve as input to optimization strategies or to provide structural alerts for oxidatively unstable compounds. As an example, Figure 7 summarizes results from FIA of a series of compounds analyzed by using EC C) Parallel EC CoulArray potentials of 0, 400, 800 and 1200 mV vs. Pd. Under these (8 channel 60 -900 mV) conditions, the products formed are generally similar to those from cytochrome P-450 catalyzed reactions that are thought to proceed through a mechanism initiated by a one- transfer oxidation. The likely reactions shown include: 1) dehydrogenation; 2) N-deacetylation; 3) 3° N-dealkylation; 4) 2° N-dealkylation; 5) S-oxidation; 6) N-oxidation; and 7) O-dealkylation.

Rapid Optimization of Formulation Antioxidants for Figure 2. LC-ESI-MS response increases significantly after post-column oxidation at a Enhanced Drug Stability coulometric electrode combined with coulometric array detection can generate the hydrodynamic voltammogram (HDV) for a compound within about 30 seconds, and is easily automated for high throughput HDV determinations. In Figure 8, experimentally determined HDVs show good agreement with oxidative stability data reported in the literature. Here, lines represent reportedly stable and dashed lines represent reportedly unstable compounds. The redox behavior described by an HDV can be used to screen oxidatively unstable candidates and rationally select potential antioxidants to include in formulation studies.5 4 Table 1. EC, ESI-MS and APCI-MS activity at pH 7 and 3.9 for neutral phenols

pH 7.0 Negative Ion pH 3.9 Positive Ion Compound Name APCI EC-Assisted ESI EC-Assisted EC-Active APCI EC-Assisted ESI EC-Assisted EC-Active Phenol Neutral Phenol 4 4 4 4 4 Catechin 4 4 4 4 Vanillin 4 4 4 4 4 Eugenol 4 4 4 4 4 4 Estradiol 4 4 4 BHA 4 4 4 4 4 4 Acetaminophen 4 4 4 4 4 2-Hydroxyestradiol 4 4 4 4 4 4 4-Hydroxyestradiol 4 4 4 4 4 4 2-Methoxyestradiol 4 4 4 4 4 4 4-Methoxyestradiol 4 4 4 4 4 4

Figure 3. On-line oxidation of tamoxifen 5

Figure 4. Effect of flow rate on % efficiency for a given cell. Oxidation efficiencies were measured for 2 µg AMI on-column (10 µL injection)

Figure 5. Precolumn oxidation of APAP with GSH as the trapping agent

Figure 6. Precolumn oxidation of phenacetin Application Note 1016

Figure 7. Representative compounds, mass shifts, and likely sites of EC oxidation

Figure 8. HDVs were generated by plotting cumulative peak area normalized with respect to the total peak area for each analyte

Conclusion References Upstream electrochemical oxidation significantly 1. Kieser, B.; Impey, G.; Meyer, D.F.; Caraiman, D.; improved MS detection for aniline, phenol and other Gamache, P. Metabolite Profiling Utilizing an In-Line compounds, produced up to microgram quantities of drug Electrochemical System for LC/MS. 52nd ASMS metabolites for further study, and detected reactive Conference on Mass Spectrometry and Allied Topics, intermediates and their conjugates too short-lived to be Nashville, TN, 2004. studied by traditional techniques such as incubation with 2. Getek, T.A.; Korfmacher, W.A.; McRae, T.A.; Hinson, liver microsomes. Drug stability could be assessed rapidly J.A. J. Chromatogr. A 1989, 474, 245-256. and the correct antioxidant(s) to be included in the 3. Deng, H. and Van Berkel, G.J. Electroanalysis 1999, formulation easily predicted. 11(12), 857-865. 4. Van Leeuwen, S.M.; Blankert, B.; Kaufmann, J-M.; Karst, U. Anal. Bioanal. Chem. 2005, 382(3), 742-750. 5. Waterman, K.C.; Adami, R.C.; Alsante, K.M.; Hong, J.; Landis, M.S.; Lombardo, F; Roberts, C.J. Pharm. Dev. Technol. 2002, 7(1), 1-32. www.thermofisher.com/dionex ©2016 Thermo Fisher Scientific Inc. All rights reserved. All trademarks are the property of Thermo Fisher Scientific Inc. and its subsidiaries. This information is presented as an example of the capabilities of Thermo Fisher Scientific Inc. products. It is not intended to encourage use of these products in any manners that might infringe the intellectual property rights of others. Specifications, terms and pricing are subject to change. Not all products are available in all countries. Please consult your local sales representative for details.

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