Electrochemistry As an Adjunct to Mass Spectrometry in Drug Development
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Application Note Application Electrochemistry as an Adjunct to Mass 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 ionization, Drug metabolites, Reactive intermediate Goal To show how on-line HPLC-electrochemical oxidation-mass spectrometry 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 Sample Preparation electrochemical nature of many drugs goes beyond simple Samples were dissolved in initial mobile phase solution to analysis. This commonality of oxidation makes EC a concentration of 20 µg/mL. particularly well suited to studying the oxidative processes in pharmaceuticals, both in the formulation and by the Liquid Chromatography 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 of drug metabolites; c) for the detection of Gradient: 4 min gradient 1% to 80% acetonitrile with constant supporting electrolyte of 20 mM ammonium acetate (pH 7) reactive intermediates and their conjugates; d) for identifi- or 50 mM formic acid 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 spectrometer 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 Electrospray ionization (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 gas: 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 temperature: 350 °C column allowed for detailed characterization of the Capillary voltage: 3500 V EC-generated products. Figure 3 shows a total ion chromatogram obtained for tamoxifen with precolumn Data Analysis EC oxidation at 1000 mV vs. Pd reference electrode. 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 solutions 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 electrolysis 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 aniline, 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 redox 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 structures leads 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 molecules. 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 lead 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