Electrochemistry as an Adjunct to Mass Spectrometry in Drug Development

John Waraska, Ian N. Acworth, David Thomas, Paul Gamache Thermo Fisher Scientific, Chelmsford, MA, USA

2 Electrochemistry as an Adjunct to Mass Spectrometry in Drug Development

Electrochemistry as an Adjunct to Mass Spectrometry in Drug Development John Waraska, Ian N. Acworth, David Thomas, Paul Gamache Thermo Fisher Scientific, Chelmsford, MA, USA

Conclusion Coulometric EC flow cells can be combined with LC/MS in a versatile platform

to rapidly study drug metabolism, toxicity and stability.

EC-assisted ionization can enable MS detection of species poorly ionized by traditional APCI or ESI.

Coulometric EC synthesis cells may be used to produce and analyze microgram quantities of oxidative metabolites on-line with a single injection.

References 1. Kieser, B.; Impey, G.; Meyer, D.F.; Caraiman, D.; Gamache, P. Metabolite Profiling Utilizing an In-Line

Electrochemical System for LC/MS. 52nd ASMS Conference on Mass Spectrometry and Allied Topics, Nashville, TN, 2004.

2. Getek, T.A.; Korfmacher, W.A.; McRae, T.A.; Hinson, J.A. J. Chromatogr. A 1989, 474, 245-256.

3. Deng, H. and Van Berkel, G.J. Electroanalysis 1999, 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.

Overview Purpose: To show how online HPLC-electrochemical oxidation-mass spectrometry can be harnessed to accelerate several key steps in drug development.

Methods: Most applications used gradient analytical or semi-preparative HPLC with pre- or post-column electrochemical oxidation followed by mass spectrometric detection. Drug stability studies used flow injection analysis with coulometric array detection.

Results: Upstream electrochemical oxidation significantly improved MS detection for aniline, phenol and other compounds, produced up to microgram quantities of drug metabolites for further study, and detected reactive intermediates and their conjugates too short-lived to be studied by traditional techniques such as incubation with liver microsomes. Drug stability could be accessed rapidly and the correct antioxidant(s) to be included in the formulation easily predicted.

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 toxicity, and is also involved in drug stability and degradation. Electrochemistry (EC) when used with HPLC has long been recognized as a sensitive and selective detection technique. Indeed more than 90% of pharmaceuticals in the market place are easily oxidized and can be detected using this technique. However, the inherent electrochemical nature of many drugs goes beyond simple analysis. This commonality of oxidation makes EC 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 and MS in a number of formats that can be used: a) to enhance ionization, thereby extending the range of compounds measured by LC-MS; b) for the micro-synthesis and identification of drug metabolites; c) for the detection of reactive intermediates and their conjugates; d) for identification of potentially problematic molecular “soft spots”; and e) to rapidly study drug stability and predict which antioxidant(s) should be included in formulations.

Methods Sample Preparation Samples were dissolved in initial mobile phase solution to a concentration of 20 µg/mL.

Liquid Chromatography HPLC: Pump, autosampler and detectors as specified below Column: CapCell® PAK C18 MG column (3 µm, 4.6 × 50 mm) Gradient: 4 min gradient 1% to 80% acetonitrile with constant supporting electrolyte of 20 mM ammonium acetate (pH 7) or 50 mM formic acid and 10 mM ammonium formate (pH 3.9), 2 mL/min Detector: Thermo Scientific Dionex Coulochem III Electrochemical Detector for MS (with model 5021A Conditioning Cell or model 5125 Synthesis Cell) or Thermo Scientific Dionex CoulArray Multi-Channel (8) Electrochemical Detector

Mass Spectrometry Single quadrupole mass spectrometer. Positive electrospray ionization (ESI), scan 80 to 500 m/z, Fragmentor 70, Gain 1.0, Threshold 150, step 0.25, drying gas 12 L/min, Nebulizer 35 psig, drying gas 3500°C, capillary voltage 3500V.

Data Analysis CoulArray™ Data Station Program 3.10.

FIGURE 1. Instrument Configuration

Results Enhanced Ionization

The experimental set-up shown in Figure 1 allows for chromatographic separation followed by a flow split. One branch of the flow undergoes electrolysis with a model 5021 conditioning cell followed by MS detection, while a parallel branch passes through a CoulArray 8-channel cell pack (sequentially higher potential per channel).

Figure 2 shows that upstream oxidation of HPLC-separated aniline, phenol, o-toluidine and BHA (500 mV, Figure 2B) led to significantly increased MS response when compared to that obtained with the EC cell off (Figure 2A).

Figure 2C shows parallel EC-Array detection of the compounds. In similar experiments, EC reactions were observed according to the following general rank order (by relative ease of oxidation) o, p-quinol and o, p-aminophenol > tertiary amine > m-quinol ≈ phenol ≈ arylamine > secondary amine ≈ thiol > thioether. No EC oxidation was observed for primary aliphatic amines and alcohols and oxidation of secondary and tertiary amines was less facile at pH 3.9, than at pH 7. These differences in oxidation potential provide a means of increased resolution and selectivity when using parallel EC-Array detection. Furthermore, differences in redox potential may also provide a means of distinguishing isobaric species observed by MS based on the use of upstream EC oxidation. This may be particularly useful for drug biotransformation studies to distinguish, for example, aromatic and aliphatic hydroxylation – oxidation of the latter being much less favorable.

Online micro-synthesis and identification of drug metabolites

Placing the coulometric oxidation cell before the HPLC column allows for detailed characterization of the EC-generated products. Figure 3 shows a total ion chromatogram obtained for tamoxifen with precolumn EC oxidation at 1000 mV vs. Pd. Several peaks corresponding to oxidation products are evident. This example illustrates on-line generation and analysis of EC products using LC-MS conditions that are typical of metabolic studies, e.g., in vitro microsomal analysis. Serial LC-EC-MS can thus be used with neat parent compound solutions for preliminary optimization of LC and MS/MS conditions and subsequent metabolite analysis in biological samples. By using identical conditions, EC data may then be used as input to automated metabolite identification software to aid in finding metabolites present in more complex biological matrices.1

CAPCELL PAK is a registered trademark of Shiseido Co. All other trademarks are the property of Thermo Fisher Scientific and its subsidiaries.

This information is not intended to encourage use of these products in any manner that might infringe the intellectual property rights of others. PO70056_E 02/12S

Table 1. EC, ESI-MS and APCI-MS activity at pH 7 and 3.9 for neutral phenols.

FIGURE 3. On-line oxidation of tamoxifen.

FIGURE 5. Precolumn oxidation of APAP with GSH as trapping agent.

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

Detection and trapping of reactive intermediates

Many studies suggest that redox metabolism of a wide range of chemical structures leads to formation of reactive 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 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 5021 cell was held at 500 mV vs. Pd.

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 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 potentials of 0, 400, 800 and 1200 mV vs. Pd. Under these conditions, the products formed are generally similar to cytochrome P-450 catalyzed reactions that are thought to proceed through a mechanism initiated by a one-electron 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; 7) O-dealkylation.

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 2. LC-ESI-MS response increases significantly after post-column oxidation at a coulometric electrode.

Furthermore, when the data from an EC-generated product correspond to that of a biological metabolite, the EC technique may then be viewed as a selective and rapid synthetic route to small quantities of this metabolite. In this example, a model 5021A conditioning cell was used to produce estimated nanogram quantities of metabolites.

Higher-capacity model 5125 cells provide the ability to produce larger quantities for more detailed structural elucidation studies. Figure 4 shows that high efficiency is maintained for oxidation of 2 µg quantities of amitriptyline (AMI) over a range of flow rates, thus facilitating the production of quantities that may be analyzed by nuclear magnetic resonance (NMR). Oxidation efficiency was calculated by subtracting the normalized AMI UV response obtained with the cell on from that obtained with the cell off (UV response with cell off = 100%).

Figure 6 shows the precolumn oxidation of phenacetin by a model 5021 cell set at 500 mV vs. Pd. The HPLC detector was an 8-channel CoulArray 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, 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 biological preparation.

FIGURE 6. Precolumn oxidation of phenacetin.

Rapid optimization of formulation antioxidants for enhanced drug stability

Flow injection analysis combined with coulometric array detection can generate the hydrodynamic voltammogram (HDV) for a compound within about 30 sec, 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, solid 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

Data from EC-assisted ionization of 11 neutral phenols compiled in Table 1 show that all four compounds that exhibit a weak ESI- or atmospheric pressure chemical ionization (APCI)-MS response at pH 7, and are EC-active, gave a large MS response after upstream EC oxidation. Similarly, all nine compounds that exhibit a weak ESI- or APCI-MS response at pH 3.9, and are EC-active, gave a large MS response after the upstream EC oxidation. EC-assisted ionization was only attempted when there was no MS response.

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

pH 7.0 Negative Ion pH 3.9 Positive IonCompound Name APCI EC-Assisted ESI EC-Assisted EC-Active APCI EC-Assisted ESI EC-Assisted EC-Active

Phenol Neutral Phenol a a a a a a

Catechin a a a aVanillin a a a a aEugenol a a a a a aEstradiol a a a

BHA a a a a a aAcetaminophen a a a a a a

2-Hydroxyestradiol a a a a a a4-Hydroxyestradiol a a a a a a2-Methoxyestradiol a a a a a a4-Methoxyestradiol a a a a a a

A

B

C

-0.3

-0.1

0.1

0.3

0.5

0.7

0.9

1.1

1.3

1.5

-300 -200 -100 0 100 200 300 400 500 600 700 800 900 1000 1100 1200 Potential (mV vs. Pd) No

rmali

zed

Cum

ulat

ive A

rea (

coul

ombs

)

Ascorbic acid Amitriptyline BHA Captopril Chlorpromazine Fluoxetine Tetracycline

3 Electrochemistry as an Adjunct to Mass Spectrometry in Drug Development

Electrochemistry as an Adjunct to Mass Spectrometry in Drug Development John Waraska, Ian N. Acworth, David Thomas, Paul Gamache Thermo Fisher Scientific, Chelmsford, MA, USA

Conclusion Coulometric EC flow cells can be combined with LC/MS in a versatile platform

to rapidly study drug metabolism, toxicity and stability.

EC-assisted ionization can enable MS detection of species poorly ionized by traditional APCI or ESI.

Coulometric EC synthesis cells may be used to produce and analyze microgram quantities of oxidative metabolites on-line with a single injection.

References 1. Kieser, B.; Impey, G.; Meyer, D.F.; Caraiman, D.; Gamache, P. Metabolite Profiling Utilizing an In-Line

Electrochemical System for LC/MS. 52nd ASMS Conference on Mass Spectrometry and Allied Topics, Nashville, TN, 2004.

2. Getek, T.A.; Korfmacher, W.A.; McRae, T.A.; Hinson, J.A. J. Chromatogr. A 1989, 474, 245-256.

3. Deng, H. and Van Berkel, G.J. Electroanalysis 1999, 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.

Overview Purpose: To show how online HPLC-electrochemical oxidation-mass spectrometry can be harnessed to accelerate several key steps in drug development.

Methods: Most applications used gradient analytical or semi-preparative HPLC with pre- or post-column electrochemical oxidation followed by mass spectrometric detection. Drug stability studies used flow injection analysis with coulometric array detection.

Results: Upstream electrochemical oxidation significantly improved MS detection for aniline, phenol and other compounds, produced up to microgram quantities of drug metabolites for further study, and detected reactive intermediates and their conjugates too short-lived to be studied by traditional techniques such as incubation with liver microsomes. Drug stability could be accessed rapidly and the correct antioxidant(s) to be included in the formulation easily predicted.

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 toxicity, and is also involved in drug stability and degradation. Electrochemistry (EC) when used with HPLC has long been recognized as a sensitive and selective detection technique. Indeed more than 90% of pharmaceuticals in the market place are easily oxidized and can be detected using this technique. However, the inherent electrochemical nature of many drugs goes beyond simple analysis. This commonality of oxidation makes EC 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 and MS in a number of formats that can be used: a) to enhance ionization, thereby extending the range of compounds measured by LC-MS; b) for the micro-synthesis and identification of drug metabolites; c) for the detection of reactive intermediates and their conjugates; d) for identification of potentially problematic molecular “soft spots”; and e) to rapidly study drug stability and predict which antioxidant(s) should be included in formulations.

Methods Sample Preparation Samples were dissolved in initial mobile phase solution to a concentration of 20 µg/mL.

Liquid Chromatography HPLC: Pump, autosampler and detectors as specified below Column: CapCell® PAK C18 MG column (3 µm, 4.6 × 50 mm) Gradient: 4 min gradient 1% to 80% acetonitrile with constant supporting electrolyte of 20 mM ammonium acetate (pH 7) or 50 mM formic acid and 10 mM ammonium formate (pH 3.9), 2 mL/min Detector: Thermo Scientific Dionex Coulochem III Electrochemical Detector for MS (with model 5021A Conditioning Cell or model 5125 Synthesis Cell) or Thermo Scientific Dionex CoulArray Multi-Channel (8) Electrochemical Detector

Mass Spectrometry Single quadrupole mass spectrometer. Positive electrospray ionization (ESI), scan 80 to 500 m/z, Fragmentor 70, Gain 1.0, Threshold 150, step 0.25, drying gas 12 L/min, Nebulizer 35 psig, drying gas 3500°C, capillary voltage 3500V.

Data Analysis CoulArray™ Data Station Program 3.10.

FIGURE 1. Instrument Configuration

Results Enhanced Ionization

The experimental set-up shown in Figure 1 allows for chromatographic separation followed by a flow split. One branch of the flow undergoes electrolysis with a model 5021 conditioning cell followed by MS detection, while a parallel branch passes through a CoulArray 8-channel cell pack (sequentially higher potential per channel).

Figure 2 shows that upstream oxidation of HPLC-separated aniline, phenol, o-toluidine and BHA (500 mV, Figure 2B) led to significantly increased MS response when compared to that obtained with the EC cell off (Figure 2A).

Figure 2C shows parallel EC-Array detection of the compounds. In similar experiments, EC reactions were observed according to the following general rank order (by relative ease of oxidation) o, p-quinol and o, p-aminophenol > tertiary amine > m-quinol ≈ phenol ≈ arylamine > secondary amine ≈ thiol > thioether. No EC oxidation was observed for primary aliphatic amines and alcohols and oxidation of secondary and tertiary amines was less facile at pH 3.9, than at pH 7. These differences in oxidation potential provide a means of increased resolution and selectivity when using parallel EC-Array detection. Furthermore, differences in redox potential may also provide a means of distinguishing isobaric species observed by MS based on the use of upstream EC oxidation. This may be particularly useful for drug biotransformation studies to distinguish, for example, aromatic and aliphatic hydroxylation – oxidation of the latter being much less favorable.

Online micro-synthesis and identification of drug metabolites

Placing the coulometric oxidation cell before the HPLC column allows for detailed characterization of the EC-generated products. Figure 3 shows a total ion chromatogram obtained for tamoxifen with precolumn EC oxidation at 1000 mV vs. Pd. Several peaks corresponding to oxidation products are evident. This example illustrates on-line generation and analysis of EC products using LC-MS conditions that are typical of metabolic studies, e.g., in vitro microsomal analysis. Serial LC-EC-MS can thus be used with neat parent compound solutions for preliminary optimization of LC and MS/MS conditions and subsequent metabolite analysis in biological samples. By using identical conditions, EC data may then be used as input to automated metabolite identification software to aid in finding metabolites present in more complex biological matrices.1

CAPCELL PAK is a registered trademark of Shiseido Co. All other trademarks are the property of Thermo Fisher Scientific and its subsidiaries.

This information is not intended to encourage use of these products in any manner that might infringe the intellectual property rights of others. PO70056_E 02/12S

Table 1. EC, ESI-MS and APCI-MS activity at pH 7 and 3.9 for neutral phenols.

FIGURE 3. On-line oxidation of tamoxifen.

FIGURE 5. Precolumn oxidation of APAP with GSH as trapping agent.

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

Detection and trapping of reactive intermediates

Many studies suggest that redox metabolism of a wide range of chemical structures leads to formation of reactive 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 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 5021 cell was held at 500 mV vs. Pd.

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 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 potentials of 0, 400, 800 and 1200 mV vs. Pd. Under these conditions, the products formed are generally similar to cytochrome P-450 catalyzed reactions that are thought to proceed through a mechanism initiated by a one-electron 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; 7) O-dealkylation.

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 2. LC-ESI-MS response increases significantly after post-column oxidation at a coulometric electrode.

Furthermore, when the data from an EC-generated product correspond to that of a biological metabolite, the EC technique may then be viewed as a selective and rapid synthetic route to small quantities of this metabolite. In this example, a model 5021A conditioning cell was used to produce estimated nanogram quantities of metabolites.

Higher-capacity model 5125 cells provide the ability to produce larger quantities for more detailed structural elucidation studies. Figure 4 shows that high efficiency is maintained for oxidation of 2 µg quantities of amitriptyline (AMI) over a range of flow rates, thus facilitating the production of quantities that may be analyzed by nuclear magnetic resonance (NMR). Oxidation efficiency was calculated by subtracting the normalized AMI UV response obtained with the cell on from that obtained with the cell off (UV response with cell off = 100%).

Figure 6 shows the precolumn oxidation of phenacetin by a model 5021 cell set at 500 mV vs. Pd. The HPLC detector was an 8-channel CoulArray 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, 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 biological preparation.

FIGURE 6. Precolumn oxidation of phenacetin.

Rapid optimization of formulation antioxidants for enhanced drug stability

Flow injection analysis combined with coulometric array detection can generate the hydrodynamic voltammogram (HDV) for a compound within about 30 sec, 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, solid 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

Data from EC-assisted ionization of 11 neutral phenols compiled in Table 1 show that all four compounds that exhibit a weak ESI- or atmospheric pressure chemical ionization (APCI)-MS response at pH 7, and are EC-active, gave a large MS response after upstream EC oxidation. Similarly, all nine compounds that exhibit a weak ESI- or APCI-MS response at pH 3.9, and are EC-active, gave a large MS response after the upstream EC oxidation. EC-assisted ionization was only attempted when there was no MS response.

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

pH 7.0 Negative Ion pH 3.9 Positive IonCompound Name APCI EC-Assisted ESI EC-Assisted EC-Active APCI EC-Assisted ESI EC-Assisted EC-Active

Phenol Neutral Phenol a a a a a a

Catechin a a a aVanillin a a a a aEugenol a a a a a aEstradiol a a a

BHA a a a a a aAcetaminophen a a a a a a

2-Hydroxyestradiol a a a a a a4-Hydroxyestradiol a a a a a a2-Methoxyestradiol a a a a a a4-Methoxyestradiol a a a a a a

A

B

C

-0.3

-0.1

0.1

0.3

0.5

0.7

0.9

1.1

1.3

1.5

-300 -200 -100 0 100 200 300 400 500 600 700 800 900 1000 1100 1200 Potential (mV vs. Pd) No

rmali

zed

Cum

ulat

ive A

rea (

coul

ombs

)

Ascorbic acid Amitriptyline BHA Captopril Chlorpromazine Fluoxetine Tetracycline

4 Electrochemistry as an Adjunct to Mass Spectrometry in Drug Development

Electrochemistry as an Adjunct to Mass Spectrometry in Drug Development John Waraska, Ian N. Acworth, David Thomas, Paul Gamache Thermo Fisher Scientific, Chelmsford, MA, USA

Conclusion Coulometric EC flow cells can be combined with LC/MS in a versatile platform

to rapidly study drug metabolism, toxicity and stability.

EC-assisted ionization can enable MS detection of species poorly ionized by traditional APCI or ESI.

Coulometric EC synthesis cells may be used to produce and analyze microgram quantities of oxidative metabolites on-line with a single injection.

References 1. Kieser, B.; Impey, G.; Meyer, D.F.; Caraiman, D.; Gamache, P. Metabolite Profiling Utilizing an In-Line

Electrochemical System for LC/MS. 52nd ASMS Conference on Mass Spectrometry and Allied Topics, Nashville, TN, 2004.

2. Getek, T.A.; Korfmacher, W.A.; McRae, T.A.; Hinson, J.A. J. Chromatogr. A 1989, 474, 245-256.

3. Deng, H. and Van Berkel, G.J. Electroanalysis 1999, 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.

Overview Purpose: To show how online HPLC-electrochemical oxidation-mass spectrometry can be harnessed to accelerate several key steps in drug development.

Methods: Most applications used gradient analytical or semi-preparative HPLC with pre- or post-column electrochemical oxidation followed by mass spectrometric detection. Drug stability studies used flow injection analysis with coulometric array detection.

Results: Upstream electrochemical oxidation significantly improved MS detection for aniline, phenol and other compounds, produced up to microgram quantities of drug metabolites for further study, and detected reactive intermediates and their conjugates too short-lived to be studied by traditional techniques such as incubation with liver microsomes. Drug stability could be accessed rapidly and the correct antioxidant(s) to be included in the formulation easily predicted.

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 toxicity, and is also involved in drug stability and degradation. Electrochemistry (EC) when used with HPLC has long been recognized as a sensitive and selective detection technique. Indeed more than 90% of pharmaceuticals in the market place are easily oxidized and can be detected using this technique. However, the inherent electrochemical nature of many drugs goes beyond simple analysis. This commonality of oxidation makes EC 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 and MS in a number of formats that can be used: a) to enhance ionization, thereby extending the range of compounds measured by LC-MS; b) for the micro-synthesis and identification of drug metabolites; c) for the detection of reactive intermediates and their conjugates; d) for identification of potentially problematic molecular “soft spots”; and e) to rapidly study drug stability and predict which antioxidant(s) should be included in formulations.

Methods Sample Preparation Samples were dissolved in initial mobile phase solution to a concentration of 20 µg/mL.

Liquid Chromatography HPLC: Pump, autosampler and detectors as specified below Column: CapCell® PAK C18 MG column (3 µm, 4.6 × 50 mm) Gradient: 4 min gradient 1% to 80% acetonitrile with constant supporting electrolyte of 20 mM ammonium acetate (pH 7) or 50 mM formic acid and 10 mM ammonium formate (pH 3.9), 2 mL/min Detector: Thermo Scientific Dionex Coulochem III Electrochemical Detector for MS (with model 5021A Conditioning Cell or model 5125 Synthesis Cell) or Thermo Scientific Dionex CoulArray Multi-Channel (8) Electrochemical Detector

Mass Spectrometry Single quadrupole mass spectrometer. Positive electrospray ionization (ESI), scan 80 to 500 m/z, Fragmentor 70, Gain 1.0, Threshold 150, step 0.25, drying gas 12 L/min, Nebulizer 35 psig, drying gas 3500°C, capillary voltage 3500V.

Data Analysis CoulArray™ Data Station Program 3.10.

FIGURE 1. Instrument Configuration

Results Enhanced Ionization

The experimental set-up shown in Figure 1 allows for chromatographic separation followed by a flow split. One branch of the flow undergoes electrolysis with a model 5021 conditioning cell followed by MS detection, while a parallel branch passes through a CoulArray 8-channel cell pack (sequentially higher potential per channel).

Figure 2 shows that upstream oxidation of HPLC-separated aniline, phenol, o-toluidine and BHA (500 mV, Figure 2B) led to significantly increased MS response when compared to that obtained with the EC cell off (Figure 2A).

Figure 2C shows parallel EC-Array detection of the compounds. In similar experiments, EC reactions were observed according to the following general rank order (by relative ease of oxidation) o, p-quinol and o, p-aminophenol > tertiary amine > m-quinol ≈ phenol ≈ arylamine > secondary amine ≈ thiol > thioether. No EC oxidation was observed for primary aliphatic amines and alcohols and oxidation of secondary and tertiary amines was less facile at pH 3.9, than at pH 7. These differences in oxidation potential provide a means of increased resolution and selectivity when using parallel EC-Array detection. Furthermore, differences in redox potential may also provide a means of distinguishing isobaric species observed by MS based on the use of upstream EC oxidation. This may be particularly useful for drug biotransformation studies to distinguish, for example, aromatic and aliphatic hydroxylation – oxidation of the latter being much less favorable.

Online micro-synthesis and identification of drug metabolites

Placing the coulometric oxidation cell before the HPLC column allows for detailed characterization of the EC-generated products. Figure 3 shows a total ion chromatogram obtained for tamoxifen with precolumn EC oxidation at 1000 mV vs. Pd. Several peaks corresponding to oxidation products are evident. This example illustrates on-line generation and analysis of EC products using LC-MS conditions that are typical of metabolic studies, e.g., in vitro microsomal analysis. Serial LC-EC-MS can thus be used with neat parent compound solutions for preliminary optimization of LC and MS/MS conditions and subsequent metabolite analysis in biological samples. By using identical conditions, EC data may then be used as input to automated metabolite identification software to aid in finding metabolites present in more complex biological matrices.1

CAPCELL PAK is a registered trademark of Shiseido Co. All other trademarks are the property of Thermo Fisher Scientific and its subsidiaries.

This information is not intended to encourage use of these products in any manner that might infringe the intellectual property rights of others. PO70056_E 02/12S

Table 1. EC, ESI-MS and APCI-MS activity at pH 7 and 3.9 for neutral phenols.

FIGURE 3. On-line oxidation of tamoxifen.

FIGURE 5. Precolumn oxidation of APAP with GSH as trapping agent.

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

Detection and trapping of reactive intermediates

Many studies suggest that redox metabolism of a wide range of chemical structures leads to formation of reactive 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 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 5021 cell was held at 500 mV vs. Pd.

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 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 potentials of 0, 400, 800 and 1200 mV vs. Pd. Under these conditions, the products formed are generally similar to cytochrome P-450 catalyzed reactions that are thought to proceed through a mechanism initiated by a one-electron 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; 7) O-dealkylation.

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 2. LC-ESI-MS response increases significantly after post-column oxidation at a coulometric electrode.

Furthermore, when the data from an EC-generated product correspond to that of a biological metabolite, the EC technique may then be viewed as a selective and rapid synthetic route to small quantities of this metabolite. In this example, a model 5021A conditioning cell was used to produce estimated nanogram quantities of metabolites.

Higher-capacity model 5125 cells provide the ability to produce larger quantities for more detailed structural elucidation studies. Figure 4 shows that high efficiency is maintained for oxidation of 2 µg quantities of amitriptyline (AMI) over a range of flow rates, thus facilitating the production of quantities that may be analyzed by nuclear magnetic resonance (NMR). Oxidation efficiency was calculated by subtracting the normalized AMI UV response obtained with the cell on from that obtained with the cell off (UV response with cell off = 100%).

Figure 6 shows the precolumn oxidation of phenacetin by a model 5021 cell set at 500 mV vs. Pd. The HPLC detector was an 8-channel CoulArray 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, 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 biological preparation.

FIGURE 6. Precolumn oxidation of phenacetin.

Rapid optimization of formulation antioxidants for enhanced drug stability

Flow injection analysis combined with coulometric array detection can generate the hydrodynamic voltammogram (HDV) for a compound within about 30 sec, 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, solid 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

Data from EC-assisted ionization of 11 neutral phenols compiled in Table 1 show that all four compounds that exhibit a weak ESI- or atmospheric pressure chemical ionization (APCI)-MS response at pH 7, and are EC-active, gave a large MS response after upstream EC oxidation. Similarly, all nine compounds that exhibit a weak ESI- or APCI-MS response at pH 3.9, and are EC-active, gave a large MS response after the upstream EC oxidation. EC-assisted ionization was only attempted when there was no MS response.

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

pH 7.0 Negative Ion pH 3.9 Positive IonCompound Name APCI EC-Assisted ESI EC-Assisted EC-Active APCI EC-Assisted ESI EC-Assisted EC-Active

Phenol Neutral Phenol a a a a a a

Catechin a a a aVanillin a a a a aEugenol a a a a a aEstradiol a a a

BHA a a a a a aAcetaminophen a a a a a a

2-Hydroxyestradiol a a a a a a4-Hydroxyestradiol a a a a a a2-Methoxyestradiol a a a a a a4-Methoxyestradiol a a a a a a

A

B

C

-0.3

-0.1

0.1

0.3

0.5

0.7

0.9

1.1

1.3

1.5

-300 -200 -100 0 100 200 300 400 500 600 700 800 900 1000 1100 1200 Potential (mV vs. Pd) No

rmali

zed

Cum

ulat

ive A

rea (

coul

ombs

)

Ascorbic acid Amitriptyline BHA Captopril Chlorpromazine Fluoxetine Tetracycline

5 Electrochemistry as an Adjunct to Mass Spectrometry in Drug Development

Electrochemistry as an Adjunct to Mass Spectrometry in Drug Development John Waraska, Ian N. Acworth, David Thomas, Paul Gamache Thermo Fisher Scientific, Chelmsford, MA, USA

Conclusion Coulometric EC flow cells can be combined with LC/MS in a versatile platform

to rapidly study drug metabolism, toxicity and stability.

EC-assisted ionization can enable MS detection of species poorly ionized by traditional APCI or ESI.

Coulometric EC synthesis cells may be used to produce and analyze microgram quantities of oxidative metabolites on-line with a single injection.

References 1. Kieser, B.; Impey, G.; Meyer, D.F.; Caraiman, D.; Gamache, P. Metabolite Profiling Utilizing an In-Line

Electrochemical System for LC/MS. 52nd ASMS Conference on Mass Spectrometry and Allied Topics, Nashville, TN, 2004.

2. Getek, T.A.; Korfmacher, W.A.; McRae, T.A.; Hinson, J.A. J. Chromatogr. A 1989, 474, 245-256.

3. Deng, H. and Van Berkel, G.J. Electroanalysis 1999, 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.

Overview Purpose: To show how online HPLC-electrochemical oxidation-mass spectrometry can be harnessed to accelerate several key steps in drug development.

Methods: Most applications used gradient analytical or semi-preparative HPLC with pre- or post-column electrochemical oxidation followed by mass spectrometric detection. Drug stability studies used flow injection analysis with coulometric array detection.

Results: Upstream electrochemical oxidation significantly improved MS detection for aniline, phenol and other compounds, produced up to microgram quantities of drug metabolites for further study, and detected reactive intermediates and their conjugates too short-lived to be studied by traditional techniques such as incubation with liver microsomes. Drug stability could be accessed rapidly and the correct antioxidant(s) to be included in the formulation easily predicted.

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 toxicity, and is also involved in drug stability and degradation. Electrochemistry (EC) when used with HPLC has long been recognized as a sensitive and selective detection technique. Indeed more than 90% of pharmaceuticals in the market place are easily oxidized and can be detected using this technique. However, the inherent electrochemical nature of many drugs goes beyond simple analysis. This commonality of oxidation makes EC 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 and MS in a number of formats that can be used: a) to enhance ionization, thereby extending the range of compounds measured by LC-MS; b) for the micro-synthesis and identification of drug metabolites; c) for the detection of reactive intermediates and their conjugates; d) for identification of potentially problematic molecular “soft spots”; and e) to rapidly study drug stability and predict which antioxidant(s) should be included in formulations.

Methods Sample Preparation Samples were dissolved in initial mobile phase solution to a concentration of 20 µg/mL.

Liquid Chromatography HPLC: Pump, autosampler and detectors as specified below Column: CapCell® PAK C18 MG column (3 µm, 4.6 × 50 mm) Gradient: 4 min gradient 1% to 80% acetonitrile with constant supporting electrolyte of 20 mM ammonium acetate (pH 7) or 50 mM formic acid and 10 mM ammonium formate (pH 3.9), 2 mL/min Detector: Thermo Scientific Dionex Coulochem III Electrochemical Detector for MS (with model 5021A Conditioning Cell or model 5125 Synthesis Cell) or Thermo Scientific Dionex CoulArray Multi-Channel (8) Electrochemical Detector

Mass Spectrometry Single quadrupole mass spectrometer. Positive electrospray ionization (ESI), scan 80 to 500 m/z, Fragmentor 70, Gain 1.0, Threshold 150, step 0.25, drying gas 12 L/min, Nebulizer 35 psig, drying gas 3500°C, capillary voltage 3500V.

Data Analysis CoulArray™ Data Station Program 3.10.

FIGURE 1. Instrument Configuration

Results Enhanced Ionization

The experimental set-up shown in Figure 1 allows for chromatographic separation followed by a flow split. One branch of the flow undergoes electrolysis with a model 5021 conditioning cell followed by MS detection, while a parallel branch passes through a CoulArray 8-channel cell pack (sequentially higher potential per channel).

Figure 2 shows that upstream oxidation of HPLC-separated aniline, phenol, o-toluidine and BHA (500 mV, Figure 2B) led to significantly increased MS response when compared to that obtained with the EC cell off (Figure 2A).

Figure 2C shows parallel EC-Array detection of the compounds. In similar experiments, EC reactions were observed according to the following general rank order (by relative ease of oxidation) o, p-quinol and o, p-aminophenol > tertiary amine > m-quinol ≈ phenol ≈ arylamine > secondary amine ≈ thiol > thioether. No EC oxidation was observed for primary aliphatic amines and alcohols and oxidation of secondary and tertiary amines was less facile at pH 3.9, than at pH 7. These differences in oxidation potential provide a means of increased resolution and selectivity when using parallel EC-Array detection. Furthermore, differences in redox potential may also provide a means of distinguishing isobaric species observed by MS based on the use of upstream EC oxidation. This may be particularly useful for drug biotransformation studies to distinguish, for example, aromatic and aliphatic hydroxylation – oxidation of the latter being much less favorable.

Online micro-synthesis and identification of drug metabolites

Placing the coulometric oxidation cell before the HPLC column allows for detailed characterization of the EC-generated products. Figure 3 shows a total ion chromatogram obtained for tamoxifen with precolumn EC oxidation at 1000 mV vs. Pd. Several peaks corresponding to oxidation products are evident. This example illustrates on-line generation and analysis of EC products using LC-MS conditions that are typical of metabolic studies, e.g., in vitro microsomal analysis. Serial LC-EC-MS can thus be used with neat parent compound solutions for preliminary optimization of LC and MS/MS conditions and subsequent metabolite analysis in biological samples. By using identical conditions, EC data may then be used as input to automated metabolite identification software to aid in finding metabolites present in more complex biological matrices.1

CAPCELL PAK is a registered trademark of Shiseido Co. All other trademarks are the property of Thermo Fisher Scientific and its subsidiaries.

This information is not intended to encourage use of these products in any manner that might infringe the intellectual property rights of others. PO70056_E 02/12S

Table 1. EC, ESI-MS and APCI-MS activity at pH 7 and 3.9 for neutral phenols.

FIGURE 3. On-line oxidation of tamoxifen.

FIGURE 5. Precolumn oxidation of APAP with GSH as trapping agent.

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

Detection and trapping of reactive intermediates

Many studies suggest that redox metabolism of a wide range of chemical structures leads to formation of reactive 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 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 5021 cell was held at 500 mV vs. Pd.

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 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 potentials of 0, 400, 800 and 1200 mV vs. Pd. Under these conditions, the products formed are generally similar to cytochrome P-450 catalyzed reactions that are thought to proceed through a mechanism initiated by a one-electron 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; 7) O-dealkylation.

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 2. LC-ESI-MS response increases significantly after post-column oxidation at a coulometric electrode.

Furthermore, when the data from an EC-generated product correspond to that of a biological metabolite, the EC technique may then be viewed as a selective and rapid synthetic route to small quantities of this metabolite. In this example, a model 5021A conditioning cell was used to produce estimated nanogram quantities of metabolites.

Higher-capacity model 5125 cells provide the ability to produce larger quantities for more detailed structural elucidation studies. Figure 4 shows that high efficiency is maintained for oxidation of 2 µg quantities of amitriptyline (AMI) over a range of flow rates, thus facilitating the production of quantities that may be analyzed by nuclear magnetic resonance (NMR). Oxidation efficiency was calculated by subtracting the normalized AMI UV response obtained with the cell on from that obtained with the cell off (UV response with cell off = 100%).

Figure 6 shows the precolumn oxidation of phenacetin by a model 5021 cell set at 500 mV vs. Pd. The HPLC detector was an 8-channel CoulArray 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, 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 biological preparation.

FIGURE 6. Precolumn oxidation of phenacetin.

Rapid optimization of formulation antioxidants for enhanced drug stability

Flow injection analysis combined with coulometric array detection can generate the hydrodynamic voltammogram (HDV) for a compound within about 30 sec, 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, solid 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

Data from EC-assisted ionization of 11 neutral phenols compiled in Table 1 show that all four compounds that exhibit a weak ESI- or atmospheric pressure chemical ionization (APCI)-MS response at pH 7, and are EC-active, gave a large MS response after upstream EC oxidation. Similarly, all nine compounds that exhibit a weak ESI- or APCI-MS response at pH 3.9, and are EC-active, gave a large MS response after the upstream EC oxidation. EC-assisted ionization was only attempted when there was no MS response.

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

pH 7.0 Negative Ion pH 3.9 Positive IonCompound Name APCI EC-Assisted ESI EC-Assisted EC-Active APCI EC-Assisted ESI EC-Assisted EC-Active

Phenol Neutral Phenol a a a a a a

Catechin a a a aVanillin a a a a aEugenol a a a a a aEstradiol a a a

BHA a a a a a aAcetaminophen a a a a a a

2-Hydroxyestradiol a a a a a a4-Hydroxyestradiol a a a a a a2-Methoxyestradiol a a a a a a4-Methoxyestradiol a a a a a a

A

B

C

-0.3

-0.1

0.1

0.3

0.5

0.7

0.9

1.1

1.3

1.5

-300 -200 -100 0 100 200 300 400 500 600 700 800 900 1000 1100 1200 Potential (mV vs. Pd) No

rmali

zed

Cum

ulat

ive A

rea (

coul

ombs

)

Ascorbic acid Amitriptyline BHA Captopril Chlorpromazine Fluoxetine Tetracycline

6 Electrochemistry as an Adjunct to Mass Spectrometry in Drug Development

Electrochemistry as an Adjunct to Mass Spectrometry in Drug Development John Waraska, Ian N. Acworth, David Thomas, Paul Gamache Thermo Fisher Scientific, Chelmsford, MA, USA

Conclusion Coulometric EC flow cells can be combined with LC/MS in a versatile platform

to rapidly study drug metabolism, toxicity and stability.

EC-assisted ionization can enable MS detection of species poorly ionized by traditional APCI or ESI.

Coulometric EC synthesis cells may be used to produce and analyze microgram quantities of oxidative metabolites on-line with a single injection.

References 1. Kieser, B.; Impey, G.; Meyer, D.F.; Caraiman, D.; Gamache, P. Metabolite Profiling Utilizing an In-Line

Electrochemical System for LC/MS. 52nd ASMS Conference on Mass Spectrometry and Allied Topics, Nashville, TN, 2004.

2. Getek, T.A.; Korfmacher, W.A.; McRae, T.A.; Hinson, J.A. J. Chromatogr. A 1989, 474, 245-256.

3. Deng, H. and Van Berkel, G.J. Electroanalysis 1999, 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.

Overview Purpose: To show how online HPLC-electrochemical oxidation-mass spectrometry can be harnessed to accelerate several key steps in drug development.

Methods: Most applications used gradient analytical or semi-preparative HPLC with pre- or post-column electrochemical oxidation followed by mass spectrometric detection. Drug stability studies used flow injection analysis with coulometric array detection.

Results: Upstream electrochemical oxidation significantly improved MS detection for aniline, phenol and other compounds, produced up to microgram quantities of drug metabolites for further study, and detected reactive intermediates and their conjugates too short-lived to be studied by traditional techniques such as incubation with liver microsomes. Drug stability could be accessed rapidly and the correct antioxidant(s) to be included in the formulation easily predicted.

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 toxicity, and is also involved in drug stability and degradation. Electrochemistry (EC) when used with HPLC has long been recognized as a sensitive and selective detection technique. Indeed more than 90% of pharmaceuticals in the market place are easily oxidized and can be detected using this technique. However, the inherent electrochemical nature of many drugs goes beyond simple analysis. This commonality of oxidation makes EC 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 and MS in a number of formats that can be used: a) to enhance ionization, thereby extending the range of compounds measured by LC-MS; b) for the micro-synthesis and identification of drug metabolites; c) for the detection of reactive intermediates and their conjugates; d) for identification of potentially problematic molecular “soft spots”; and e) to rapidly study drug stability and predict which antioxidant(s) should be included in formulations.

Methods Sample Preparation Samples were dissolved in initial mobile phase solution to a concentration of 20 µg/mL.

Liquid Chromatography HPLC: Pump, autosampler and detectors as specified below Column: CapCell® PAK C18 MG column (3 µm, 4.6 × 50 mm) Gradient: 4 min gradient 1% to 80% acetonitrile with constant supporting electrolyte of 20 mM ammonium acetate (pH 7) or 50 mM formic acid and 10 mM ammonium formate (pH 3.9), 2 mL/min Detector: Thermo Scientific Dionex Coulochem III Electrochemical Detector for MS (with model 5021A Conditioning Cell or model 5125 Synthesis Cell) or Thermo Scientific Dionex CoulArray Multi-Channel (8) Electrochemical Detector

Mass Spectrometry Single quadrupole mass spectrometer. Positive electrospray ionization (ESI), scan 80 to 500 m/z, Fragmentor 70, Gain 1.0, Threshold 150, step 0.25, drying gas 12 L/min, Nebulizer 35 psig, drying gas 3500°C, capillary voltage 3500V.

Data Analysis CoulArray™ Data Station Program 3.10.

FIGURE 1. Instrument Configuration

Results Enhanced Ionization

The experimental set-up shown in Figure 1 allows for chromatographic separation followed by a flow split. One branch of the flow undergoes electrolysis with a model 5021 conditioning cell followed by MS detection, while a parallel branch passes through a CoulArray 8-channel cell pack (sequentially higher potential per channel).

Figure 2 shows that upstream oxidation of HPLC-separated aniline, phenol, o-toluidine and BHA (500 mV, Figure 2B) led to significantly increased MS response when compared to that obtained with the EC cell off (Figure 2A).

Figure 2C shows parallel EC-Array detection of the compounds. In similar experiments, EC reactions were observed according to the following general rank order (by relative ease of oxidation) o, p-quinol and o, p-aminophenol > tertiary amine > m-quinol ≈ phenol ≈ arylamine > secondary amine ≈ thiol > thioether. No EC oxidation was observed for primary aliphatic amines and alcohols and oxidation of secondary and tertiary amines was less facile at pH 3.9, than at pH 7. These differences in oxidation potential provide a means of increased resolution and selectivity when using parallel EC-Array detection. Furthermore, differences in redox potential may also provide a means of distinguishing isobaric species observed by MS based on the use of upstream EC oxidation. This may be particularly useful for drug biotransformation studies to distinguish, for example, aromatic and aliphatic hydroxylation – oxidation of the latter being much less favorable.

Online micro-synthesis and identification of drug metabolites

Placing the coulometric oxidation cell before the HPLC column allows for detailed characterization of the EC-generated products. Figure 3 shows a total ion chromatogram obtained for tamoxifen with precolumn EC oxidation at 1000 mV vs. Pd. Several peaks corresponding to oxidation products are evident. This example illustrates on-line generation and analysis of EC products using LC-MS conditions that are typical of metabolic studies, e.g., in vitro microsomal analysis. Serial LC-EC-MS can thus be used with neat parent compound solutions for preliminary optimization of LC and MS/MS conditions and subsequent metabolite analysis in biological samples. By using identical conditions, EC data may then be used as input to automated metabolite identification software to aid in finding metabolites present in more complex biological matrices.1

CAPCELL PAK is a registered trademark of Shiseido Co. All other trademarks are the property of Thermo Fisher Scientific and its subsidiaries.

This information is not intended to encourage use of these products in any manner that might infringe the intellectual property rights of others. PO70056_E 02/12S

Table 1. EC, ESI-MS and APCI-MS activity at pH 7 and 3.9 for neutral phenols.

FIGURE 3. On-line oxidation of tamoxifen.

FIGURE 5. Precolumn oxidation of APAP with GSH as trapping agent.

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

Detection and trapping of reactive intermediates

Many studies suggest that redox metabolism of a wide range of chemical structures leads to formation of reactive 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 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 5021 cell was held at 500 mV vs. Pd.

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 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 potentials of 0, 400, 800 and 1200 mV vs. Pd. Under these conditions, the products formed are generally similar to cytochrome P-450 catalyzed reactions that are thought to proceed through a mechanism initiated by a one-electron 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; 7) O-dealkylation.

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 2. LC-ESI-MS response increases significantly after post-column oxidation at a coulometric electrode.

Furthermore, when the data from an EC-generated product correspond to that of a biological metabolite, the EC technique may then be viewed as a selective and rapid synthetic route to small quantities of this metabolite. In this example, a model 5021A conditioning cell was used to produce estimated nanogram quantities of metabolites.

Higher-capacity model 5125 cells provide the ability to produce larger quantities for more detailed structural elucidation studies. Figure 4 shows that high efficiency is maintained for oxidation of 2 µg quantities of amitriptyline (AMI) over a range of flow rates, thus facilitating the production of quantities that may be analyzed by nuclear magnetic resonance (NMR). Oxidation efficiency was calculated by subtracting the normalized AMI UV response obtained with the cell on from that obtained with the cell off (UV response with cell off = 100%).

Figure 6 shows the precolumn oxidation of phenacetin by a model 5021 cell set at 500 mV vs. Pd. The HPLC detector was an 8-channel CoulArray 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, 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 biological preparation.

FIGURE 6. Precolumn oxidation of phenacetin.

Rapid optimization of formulation antioxidants for enhanced drug stability

Flow injection analysis combined with coulometric array detection can generate the hydrodynamic voltammogram (HDV) for a compound within about 30 sec, 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, solid 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

Data from EC-assisted ionization of 11 neutral phenols compiled in Table 1 show that all four compounds that exhibit a weak ESI- or atmospheric pressure chemical ionization (APCI)-MS response at pH 7, and are EC-active, gave a large MS response after upstream EC oxidation. Similarly, all nine compounds that exhibit a weak ESI- or APCI-MS response at pH 3.9, and are EC-active, gave a large MS response after the upstream EC oxidation. EC-assisted ionization was only attempted when there was no MS response.

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

pH 7.0 Negative Ion pH 3.9 Positive IonCompound Name APCI EC-Assisted ESI EC-Assisted EC-Active APCI EC-Assisted ESI EC-Assisted EC-Active

Phenol Neutral Phenol a a a a a a

Catechin a a a aVanillin a a a a aEugenol a a a a a aEstradiol a a a

BHA a a a a a aAcetaminophen a a a a a a

2-Hydroxyestradiol a a a a a a4-Hydroxyestradiol a a a a a a2-Methoxyestradiol a a a a a a4-Methoxyestradiol a a a a a a

A

B

C

-0.3

-0.1

0.1

0.3

0.5

0.7

0.9

1.1

1.3

1.5

-300 -200 -100 0 100 200 300 400 500 600 700 800 900 1000 1100 1200 Potential (mV vs. Pd) No

rmali

zed

Cum

ulat

ive A

rea (

coul

ombs

)

Ascorbic acid Amitriptyline BHA Captopril Chlorpromazine Fluoxetine Tetracycline

CAPCELL PAK is a registered trademark of Shiseido Co. All other trademarks are the property of Thermo Fisher Scientific Inc. and its subsidiaries. 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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