Identifying Lactone Hydrolysis in Pharmaceuticals. A Tool for

NMR was ultimately required to characterize this compound because the MS data could not conclusively identify the structure.23 While NMR is clearly ef...
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Anal. Chem. 2005, 77, 6655-6663

Identifying Lactone Hydrolysis in Pharmaceuticals. A Tool for Metabolite Structural Characterization Lin Yi, Mary L. Bandu, and Heather Desaire*

Department of Chemistry, University of Kansas, Lawrence, Kansas 66045

Methods to characterize metabolic transformations in a rapid and reliable fashion are required for facilitating the development of all new pharmaceuticals. One metabolic transformation, which is the focus of this study, is lactone hydrolysis. For pharmaceuticals containing lactones, hydrolysis occurs readily due to both enzymatic and nonenzymatic processes. Hydrolysis affects both the bioavailability and the efficacy of lactone-containing drugs and pro-drugs. To facilitate the characterization of lactones and their corresponding hydrolysis products, we have developed a mass spectrometric method that can readily discriminate between a lactone and its corresponding carboxylic acid, even when these changes are accompanied by other modifications that occur during metabolism. This method uses characteristic product ions in MS/MS experiments, and the trends described herein can be applied broadly to several types of lactones. To demonstrate the efficacy of this approach, two different lactones that had undergone multiple modifications were characterized, and in both cases, lactone hydrolysis was readily discernible, based on the MS/MS data. Characterizing metabolic transformations of pharmaceuticals is an extremely important step in developing safe and effective drugs.1-4 Because drugs can undergo significant biotransformations and these changes alter the desired effect of the drug, metabolism studies are now being completed at early stages in the drug-development cycle.2,4 While early-stage metabolism studies are an attractive drug-development approach because they eliminate many potentially unsafe or ineffective drug candidates early in development, they can be costly.2,4 In early-stage metabolism studies, many potential drug candidates are tested, and all the metabolic products from the drug candidate pool need to be characterized and evaluated. This paradigm shift results in a pressing need for high-throughput approaches for identifying * Corresponding author. Phone: (785)864-3015. E-mail: [email protected]. (1) Gibson, G. G.; Skett, P. Introduction to Drug Metabolism; Chapman and Hall: New York, 1986. (2) Welling, P. G. Changes in pharmacokinetics and drug metabolism responsibility in drug discovery and development. In The Drug Development Process: Increasing Efficiency and Cost-Effectiveness; Welling, P. G., Lasagna, L., Banakar, U. V., Eds.; Drugs and the Pharmaceutical Sciences Vol. 76; Marcel Dekker: New York, 1996; Chapter 9. (3) Sneader, W. Production and formulation. In Drug Development: From laboratory to clinic; John Wiley & Sons: Chichester, 1986; Chapter 2. (4) Lin, J. H.; Lu, A. Y. H. Pharmacol. Rev. 1997, 49, 403-449. 10.1021/ac0507237 CCC: $30.25 Published on Web 09/14/2005

© 2005 American Chemical Society

metabolic transformations.4,5 To address this need, we are developing methods to rapidly identify common biotransformations that have a significant impact on drug efficacy and safety. One example of an important metabolic transformation is lactone hydrolysis.6-10

Many drug candidates contain this functionality, and their efficacy is directly affected by this transformation. Some examples include camptothecin, its analogues, and statins. Camptothecin analogues, which are widely used anticancer agents, are hydrolyzed to form inactive carboxylic acids under physiological conditions. This ultimately results in a compromised pharmacological effect.11-13 On the other hand, statins, which are a type of cholesterol-lowering drug, are hydrolyzed to the carboxylic acid forms to produce the desired pharmacological effect.7-9 In either case, the overall efficacy of the active pharmaceutical is highly dependent on hydrolysis of lactone ring. To investigate the metabolic process of these and similar pharmaceutical lactones, it is therefore necessary to identify the hydrolysis of lactone ring and make a clear differentiation between the lactone and the carboxylic acid form. Since these compounds can also undergo other metabolic transformations (in addition to lactone hydrolysis), the method used to monitor hydrolysis should be general enough that it can differentiate between the lactone and carboxylic acid forms of the drug, even if other metabolic changes have occurred. At present, there are many methods that can monitor the presence of a lactone and its hydrolysis products, provided that a (5) Clarke, N. J.; Rindgen, D.; Korfmacher, W. A.; Cox, K. A. Anal. Chem. 2001, 73, 430A-439A. (6) Teiber, J. F.; Draganov, D. I.; La Du, B. N. Biochem. Pharmacol. 2003, 66, 887-896. (7) Duggan, D. E.; Chen, I.-W.; Bayne, W. F.; Halpin, R. A.; Duncan, C. A.; Schwartz, M. S.; Stubbs, R. J.; Vickers, S. Drug Matab. Dispos. 1989, 17, 166-173. (8) Vickers, S.; Duncan, C. A.; Chen, I.-W.; Rosegay, A.; Duncan D. E. Drug Matab. Dispos. 1990, 18, 138-145. (9) Hamelin, B. A.; Turgeon, J. Trends Pharmacol. Sci. 1998, 19, 26-37. (10) Jacobsen, W.; Kuhn, B.; Soldner, A.; Kirchner, G.; Sewing, K.-F.; Kollma, P. A.; Benet L. Z.; Christians, U. Drug Metab. Dispos. 2000, 28, 1369-1378. (11) Mi, Z.; Burke, T. G. Biochemistry 1994, 33, 10325-10366. (12) Mi Z.; Burke T. G. Biochemistry 1994, 33, 12540-12545. (13) Rivory, L. P.; Robert J. Pharmacol. Ther. 1995, 68, 269-296.

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standard of each compound is available and that no other modifications are occurring in addition to lactone hydrolysis. Some examples include ultraviolet spectrometry (UV-Vis) and fluorometry,14 high-performance liquid chromatography (HPLC) with ultraviolet15-17 or fluorescence detection,11-12,18-20 and mass spectrometry.21-25 Although the above methods are useful for discriminating between the lactone and acid forms when no other transformations are occurring, the methods cannot be generally applied to identifying hydrolysis in early-stage metabolism studies. UV-Vis and fluorometry are very nonselective methods, and they cannot be used to identify new metabolites. Mass spectrometry is certainly a more selective method. When lactones become hydrolyzed, their mass increases by 18 Da, a change that is readily detected in MS experiments. Therefore, lactone hydrolysis could be discriminated from other modifications using MS. However, when hydrolysis occurs in conjunction with other transformations, the mass of the hydrolyzed product will not be 18 Da greater than the original lactone, so this approach is ultimately unreliable. One example of this common problem involves the pharmaceutical etamycin. This antibiotic undergoes two transformations, lactone hydrolysis and elimination of water, to produce an inactive drug in vivo. NMR was ultimately required to characterize this compound because the MS data could not conclusively identify the structure.23 While NMR is clearly effective for characterizing structural changes such as lactone hydrolysis, it is not a highthroughput approach, which limits its usability for early-stage metabolism studies. If one could rapidly identify lactone hydrolysis in unknown compounds, where hydrolysis occurred in conjunction with other modifications, such a method would greatly facilitate metabolism studies of lactone-containing pharmaceutical candidates and prodrugs such as camptothecin anologues, statins, and etamycin. Since mass spectrometry affords low detection limits and rapid data acquisition and analysis times, we have developed a rapid method of identifying lactone hydrolysis using characteristic fragmentation patterns in MS/MS data. This characterization method is applicable to metabolites of lactone-containing phar(14) Chourpa, I.; Mollot J.-M.; Sockalingum, G. D.; Riou J.-F.; Manfait, M. Biochim. Biophys. Acta 1998, 1379, 353-366. (15) Billecke, S.; Draganov, D.; Counsell, R.; Stetson, P.; Watson, C.; Hsu C.; La Du, B. N. Drug Metab. Dispos. 2000, 28, 1335-1342. (16) Lesueur-Ginot, L.; Demarquay, D.; Kiss, R.; Kasprzyk, P. G.; Dassonneville, L.; Bailly, C.; Camara, J.; Lavergne, O.; Bigg D. C. H. Cancer Res. 1999, 59, 2939-2943. (17) Carlucci, G.; Mazzeo, P.; Biordi, L.; Bologna, M. J. Pharm. Biomed. Anal. 1992, 10, 693-697. (18) Prijovich, Z. M.; Leu, Y.-L.; Roffler, S. R. Biochem. Pharmacol. 2003, 66, 1181-1187. (19) Bom, D.; Curran, D. P.; Zhang, J.; Zimmer, S. G.; Bevins, R.; Kruszewski, S.; Howe, J. N.; Bingcang, A.; Latus, L. J.; Burke, T. G. J. Controlled Release 2001, 74, 325-333. (20) Ochiai, H.; Uchiyama, N.; Imagaki, K.; Hata, S.; Kamei, T. J. Chromatogr. B 1997, 694, 211-217. (21) Takano, T.; Abe, S.; Hata, S. Biomed. Environ. Mass Spectrom. 1990, 19, 577-581. (22) Morris, M. J.; Gilbert, J. D.; Hsieh, J. Y.-K.; Matuszewski, B. K.; Ramjit, H. G.; Banye, W. F. Biol. Mass Spectrom. 1993, 22, 1-8. (23) Bateman, K. P.; Yang, K.; Thibault, P.; White, R. L.; Vining, L. C J. Am. Chem. Soc. 1996, 118, 5335-5338. (24) Jemal, M.; Rao, S.; Salahudeen, I.; Chen, B.-C.; Kates, R. J. Chromatogr. B 1999, 736, 19-41. (25) Jemal, M.; Ouyang, Z.; Powell, M. L. J. Pharm. Biomed. Anal. 2000, 23, 323-340.

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maceuticals that have been previously detected, but not characterized structurally. In this study, a group of selected pharmaceutical lactones and their corresponding carboxylic acid forms were subjected to electrospray ionization tandem mass spectrometry (ESI-MS/MS). The data obtained demonstrate that characteristic neutral losses can be utilized to differentiate between lactone and acid forms, and these characteristic losses can be directly attributed to the site of lactone hydrolysis. Mechanisms that account for the neutral losses observed are provided, and they can be used to explain why the acids and lactones dissociate differently. Using this strategy, lactone hydrolysis products and their corresponding lactones can be discriminated when specific structural features are possessed by lactones and their acids; this is addressed in detail in this paper. In the strategy presented herein, MS/MS data, not the mass of the precursor ion, are used to distinguish lactones from their acid forms. As a result, lactone hydrolysis products occurring in conjunction with other chemical modifications can be identified. This is demonstrated with two pharmacologically relevant examples. EXPERIMENTAL SECTION Stock solution of selected lactones was prepared by dissolving solid compounds in HPLC grade methanol to the concentration of 1.0 × 10-2 M. Camptothecin, SN-38, and topotecan, which were not soluble in methanol, were dissolved in DMSO to the same concentration. Nonhydrolyzed samples were prepared by directly diluting the stock solution to 1.0 × 10-4 M with methanol. Hydrolyzed samples were generated by dilution of the stock solution in 10% NH3‚H2O/90% H2O to 1.0 × 10-3 M (pH 10) and incubation during 24 h at 60 °C, followed by a further dilution to 1.0 × 10-4 M with methanol. Samples were introduced into the mass spectrometer by directly infusing the sample via a syringe pump. All the samples were analyzed on an LCQ Advantage, a quadrupole ion trap mass spectrometer (Thermo, San Jose, CA). ESI-MS/MS spectra of lactones and their acid forms were obtained in both positive and negative modes. For all the MS/MS data shown herein, identical ion activation conditions were used: The precursor ion isolation width was 5 Da, all the precursor ions were activated for 30 ms with 35% normalized collision energy (as defined by the Xcalibur 1.3 software), and the qz value was 0.30. The HPLC system used in the study was a Shimadzu LC10ATvp with a 20 µL loop and a Shimadzu SPD-M10Avp UV-Vis detector (Shimadzu, Kyoto, Japan). Separation was accomplished on a 15 cm Prevail C18 column with 4.6 mm i.d. and 5 µm particle size (Altech, Deerfield, IL). The mobile phase was 100% HPLC grade acetonitrile with a flow rate of 0.6 mL/min. The UV detector was set at 309 nm. The injected sample was in 2% NH3‚H2O/98% H2O with a concentration of 1.0 × 10-3 M. Eluents from HPLC were collected and directly introduced into mass spectrometer. RESULTS AND DISCUSSION Obtaining MS/MS Data of Lactones and Their Corresponding Carboxylic Acids. The lactones that were subjected to this analysis are shown in Table 1. This group of compounds is representative of a wide variety of lactone structures. Many of

Table 1. Characteristic Neutral Losses of Lactones and Their Acid Forms in MS/MSa

a A threshold of 5% is used for the percentage of neutral losses. “-” means no neutral loss or loss of intensity below 5%. The specific neutral losses that can help to differentiate lactone and acid forms of specific compounds are emphasized. “L” represents lactone form; “A” represents acid form. An asterisk “*” indicates that the lactone and acid forms of 11 and 12 can be differentiated in MS3, as described in the text.

the compounds have diverse functional groups, and most of them are pharmacologically relevant. The MS/MS data for the lactones were obtained by analyzing the samples in positive and negative ion modes. In this experiment, the hydrolyzed forms of the lactones were generated using base-catalyzed hydrolysis. This is a standard method used to generate the carboxylic acid forms

from the lactones.14,26,27 MS/MS data for these hydrolyzed species were also obtained in the positive and negative ion mode. The data for the lactone and acid forms are compared in Table 1. The ionization mode that can be used for MS analysis of these species is dependent upon the chemical structure. For example, the lactones that do not have protons attached to heteroatoms do Analytical Chemistry, Vol. 77, No. 20, October 15, 2005

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not ionize readily in negative ion mode. For this set of lactones (3, 4, 5, 7, 10, and 12), only MS/MS data in the positive ion mode are available. For lactones that have protonated heteroatoms, both positive and negative mode data are available, so both are acquired. Differentiation of Lactone and Acid Forms by Characteristic Neutral Losses. In Table 1, a limited set of diagnostic neutral losses is used to differentiate the lactones from the acidic hydrolysis products. The neutral losses were chosen based on two criteria: (1) Past literature precedence indicates that these losses can be directly attributed to fragmentation of lactones or carboxylic acids.28-33 (2) The losses are useful in distinguishing the lactones from their corresponding carboxylic acids. In negative ion mode, a loss of CO2 (44 Da) is commonly observed for many types of acids,28 losses of H2O (18 Da) and H2O + CO2 (62 Da) also originate from carboxylic acid groups.29 In positive mode, lactone fragmentation has been studied extensively. Common neutral losses include H2O (18 Da),30-33 CO (28 Da),30,31,33 and H2O + CO (46 Da).31-33 By focusing on these particular neutral losses, which originate from the part of the molecule that is changing during hydrolysis, any differences in the presence or absence of these ions in MS/MS data can be directly attributed to differences in chemical structure between the lactone and its corresponding carboxylic acid. Since this method is designed to identify hydrolysis products when more than one modification has occurred in the molecule, it is absolutely essential to use diagnostic ions that can be specifically attributed to the hydrolysis of the lactone. For lactones in Table 1, the diagnostic differences (in bold) can be used to differentiate lactones from their acid forms. The mechanisms describing these distinguishing ions are rationalized in Figures 1-4. Type 1: Differentiation by 62 Da Loss in Negative Mode. Lactones 1 and 2 can be distinguished from their acid forms because only the acids show a loss of 62 Da in (-)ESI-MS/MS data. This loss is explained in Figure 1. When the lactone undergoes a loss of 44 Da (CO2), the negative charge is resonance stabilized, and no additional fragmentation forms. The acid form dissociates differently. It undergoes the same neutral loss of 44 Da, but this loss can occur in conjunction with another loss of 18 Da (H2O) to form a more stable, conjugated product ion. When this occurs, the net loss is 62 Da, and this unique product ion discriminates the acid form from the lactone form. For this loss of 62 Da to discriminate between the lactone and acid forms, several structural features are required (see Figure 1). The lactone needs to be in a six-membered ring that contains a hydroxyl group on the R carbon. Also, the double bond between the β and γ carbons is required to drive the elimination of water (26) Chourpa, I.; Beljebbar, A.; Sockalingum, G. D.; Riou, J.-F.; Manfait, M. Biochim. Biophys. Acta 1997, 1334, 349-360. (27) Kearney, A. S.; Crawford, L. F.; Mehta, S. C.; Radebaugh, G. W. Pharm. Res. 1993, 10, 1461-1465. (28) Bandu, M. L.; Watkins, K. R.; Bretthauer, M. L.; Moore, C. A.; Desaire, H. Anal. Chem. 2004, 76, 1746-1753. (29) Kerwin, J. L.; Torvik, J. J. Anal. Biochem. 1996, 237, 56-64. (30) Tian, Q.; Kent, K. D.; Bomser, J. A.; Schwartz, S. J. Rapid Commun. Mass Spectrom. 2004, 18, 3099-3104. (31) Crotti, A. E. M.; Fonseca, T.; Hong, H.; Staunton, J.; Galembeck, S. E.; Lopes, N. P.; Gates, P. J. Int. J. Mass Spectrom. 2004, 232, 271-276. (32) Ashley, G. W.; Carney, J. R. J. Antibiot. 2004, 57, 224-234. (33) Donovan, T.; Brodbelt, J. J. Am. Soc. Mass Spectrom. 1992, 3, 47-59.

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Figure 1. Mechanism explaining the differentiation of the acid and lactone forms of 1 and 2 in (-)ESI-MS/MS. (a) The 44 Da (CO2) loss can occur from the lactone form; (b) 62 Da (CO2 + H2O) loss exclusively occurs from the acid form.

Figure 2. Mechanism explaining why the neutral loss of 18 Da (H2O) in (+)ESI-MS/MS can be used to differentiate the lactones from the corresponding acids. (a) 18 Da (H2O) loss from the acid form; (b) no 18 Da (H2O) loss from the lactone.

from the acid form. When these structural features are not present, as exemplified in 3-12, this neutral loss does not distinguish the lactone from the acid form. Type 2: Differentiation by 18 Da Loss in Positive Mode. Figure 2 demonstrates the mechanism of differentiation between lactones and their acid forms by neutral loss of 18 Da (H2O) in (+)ESIMS/MS. From Table 1, it is observed that all acid forms undergo the neutral loss of H2O, and a mechanism explaining this loss is illustrated in Figure 2a, which demonstrates that the positive charge can be resonance stabilized after the neutral H2O loss. As to the lactone forms, the results are more complicated. For the lactones containing an additional hydroxyl group, the neutral loss of H2O is likely to occur, as demonstrated by 8, 9, and 11. However, neutral loss of H2O is not observed for the lactone forms of 1 and 2, which also contain a hydroxyl group. Various structural features can affect fragmentation pathway of neutral H2O loss. Since all the acid forms can undergo this neutral loss, peaks representing this neutral loss can be used to differentiate the lactone and acid forms, if the same neutral loss is not available from lactone form. Essentially, any lactone that does not undergo loss of H2O during MS/MS could potentially be discriminated from its acid form, using this characteristic neutral loss. Compounds 1-5 are good examples. One potential caveat regarding this identification strategy is that a lactone that does not undergo loss of water may transform into a new metabolite that does produce a water loss. If this occurs, this new transformation product could not be discriminated from lactone hydrolysis using only this characteristic neutral loss. Type 3: Differentiation by 28 Da Loss in Positive Mode. In Figure 3, the mechanism that describes neutral loss of 28 Da (CO) in (+)ESI-MS/MS is illustrated. No carboxylic acids undergo neutral

Figure 3. Mechanism explaining why the neutral loss of 28 Da (CO) in (+)ESI-MS/MS can be used to differentiate the lactones from their acid forms. (a) No 28 Da (CO) loss occurs for the acid form; (b) 28 Da loss from lactone form occurs, when conditions are favorable.

loss of 28 Da (see Table 1). This is due to the fact that the acid forms can only undergo CO loss in conjunction with H2O loss. They do not undergo the CO loss independently. This is addressed in the next section. As to the lactone forms, to facilitate this neutral loss, several conditions are required, as described in Figure 3b. First, protonation on the lactone ring oxygen needs to be favorable. Therefore, lactones with other strong basic sites, like tertiary amines (1, 2, 5, and 12) do not meet this requirement, so they do not undergo the loss. Second, an R hydroxyl group or other electron-donating source is needed to stabilize R carbon. Both requirements can be met by 6-8. All three of these lactones undergo loss of CO (28 Da) during MS/MS, so all three of these lactones can be differentiated from their acid forms by the CO neutral loss. No other lactones can meet both of these requirements, as a result, their two forms cannot be differentiated by this neutral loss. In this case the general rule is as follows: If a lactone has an R hydroxy group and it does not contain an amine, it will selectively undergo a neutral loss of 28 Da during MS/MS. Other lactones, in addition to those containing an R hydroxy group, could also undergo this loss and be discriminated from their corresponding carboxylic acids, if their chemical structure allows for donation of electrons onto the R carbon. Type 4: Differentiation by 46 Da Loss in Positive Mode. Figure 4 shows the mechanism of differentiating the lactone form from its acid form by the neutral loss of 46 Da (CO + H2O) in (+)ESI-MS/MS. This loss can occur for carboxylic acids if the resulting product, which is a carbocation, is stabilized. Since this mechanism requires that the carboxylic acid be protonated, compounds with amine groups, which are substantially more basic, do not undergo this loss. In Figure 4b, one type of structure that can undergo this neutral loss is demonstrated. In this case, the positive charge can be resonance stabilized by the conjugated π system, which is one carbon away. This structure is a partial representation of 4. Figure 4c is another example of a compound that undergoes a 46 Da loss from acid form. In this case, the

Figure 4. Mechanism explaining why the neutral loss of 46 Da (CO + H2O) in (+)ESI-MS/MS can be used to differentiate lactones from their acid forms. (a) No 46 Da loss occurs for most lactones; (b-d) three examples of when the 46 Da (CO + H2O) loss occurs for carboxylic acids.

Figure 5. Mechanism explaining the neutral loss of 46 Da (CO + H2O) from the lactone form of δ-valerolactone (10). This represents an unusual case where a lactone undergoes a 46 Da loss during MS/MS.

positive charge is stabilized via a hydride shift, which makes the positive charge resonance stabilized.28 This shows a partial representation of 9. Figure 4d, which represents the fragmentation of the acid form of 8, demonstrates that, when an R hydroxy group is present, resonance stabilization of the positive charge can be obtained, so R hydroxy acids also undergo a 46 Da loss in positive mode. Since lactones rarely undergo the loss of 46 Da, several types of lactones can be differentiated by this 46 Da loss. It is important to note with this differentiation strategy that one lactone underwent a loss of 46 Da in this study. It was 10. Donovan and Brodbelt have reported that for small, structurally simple lactones, like 10, the neutral loss of 46 Da occurs by a mechanism shown in Figure 5.33 One of the requirements of this process is that β carbon of the ring oxygen needs to be sp3 hybridized, and it must contain a β hydrogen. This requirement prohibits many other structurally simple, small lactones, like 7 and 8, from undergoing this process. Larger lactones, with more diverse functional groups, are also unlikely to undergo this process, even if their β carbon is sp3 hybridized, due to the competition of other more favorable fragmentation pathways. To summarize, the loss of 46 Da occurs for carboxylic acids if the resulting product ion is stabilized. However, the mechanism requires that the carboxylic acid is protonated, so compounds containing nitrogen generally did not undergo this loss. Carboxylic acids that have the correct structural features to undergo the 46 Da neutral loss could be distinguished from their corresponding lactones because lactones generally do not undergo this loss, with one well-characterized exception. Differentiation by Neutral Losses in MS3 Experiments. Two of the 12 compounds were not readily distinguished in MS2 Analytical Chemistry, Vol. 77, No. 20, October 15, 2005

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Figure 6. Fragmentation observed in MS3 experiments. (a) Hydrastin (12) in (+)ESI-MS3; (b) andographolide (11) in (-)ESI-MS3.

Figure 7. (-)ESI-MS/MS data for topotecan: (a) lactone form; (b) acid form.

experiments, 11 and 12. These two compounds do not meet any of the structural requirements described in Figures 1-4, so the MS/MS method described above is not useful at differentiating these compounds. However, these species are readily distinguished by an MS3 experiment when the peak that corresponds to loss of water is collisionally activated. As shown in Table 1, the lactone form of 12 cannot be readily deprotonated in negative mode, so MS3 of 12 is obtained in positive mode. For 11, the signal is stronger in negative ion mode, so (-)ESI-MS3 is performed for 11. For both 11 and 12, a loss of CO is observed for the lactone form, but not the acid, during the MS3 experiment. This loss can be explained using a mechanistic rationale, as shown in Figure 6. The results indicate that MS3 is a good strategy to use if the lactone and acid forms do not meet the structural criteria described above (and therefore, cannot be discriminated by the neutral losses already mentioned). By comparing the MSn data for the lactone and carboxylic acid pairs, it is possible to distinguish all the lactones in this study from the acids. Furthermore, by focusing on neutral losses that are directly attributable to the functional groups of interest, logical 6660

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mechanistic rationales can be proposed that explain why the spectra are distinguishable. These mechanistic rationales help identify the structural features that are necessary to observe the characteristic neutral losses. Using this approach, it is possible to identify lactone hydrolysis in a wide variety of species, even when other structural modifications are present. To demonstrate the utility of this approach, two examples are provided below. Application in Pharmaceutically Relevant Examples. Example 1: Camptothecin Analogues and Metabolites. Compounds 1 and 2 in Table 1 are two structural analogues of camptothecin. In Figure 1, a mechanistic rationale was proposed that explains why the lactone forms of these two analogues were discriminated from the acid forms. On the basis of this mechanism, one could predict that any camptothecin analogue could be subjected to this MS/MS experiment, and the data could be used to readily distinguish the lactone form from the carboxylic acid. As a result, lactone hydrolysis could be monitored, even when accompanied by other structural modifications. To demonstrate that this assertion is correct, another camptothecin analogue (topotecan) was tested after the general rule in Figure 1 was developed.

Figure 8. (a) HPLC chromatogram of noscapine (5), after base hydrolysis; (b) (+)ESI-MS data for peak 1; (c) (+)ESI-MS/MS of m/z 432 (from peak 1); (d) (+)ESI-MS/MS of m/z 414 (from peak 1); (e) (+)ESI-MS data for peak 2; (f) (+)ESI-MS/MS of m/z 414 (from peak 2).

The (-)ESI-MS/MS spectra for both lactone and acid forms of topotecan are in Figure 7. For the lactone form, only a 44 Da

(CO2) loss is present. For the acid form, relatively weak peak corresponding to the 44 Da loss is present along with a strong Analytical Chemistry, Vol. 77, No. 20, October 15, 2005

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Figure 9. Mechanism for the formation of the isomer of noscapine.

peak corresponding to a 62 Da (CO2 and H2O) loss. The net 62 Da loss from the acid form can thus differentiate the two forms, and this discriminating feature was predicted by the mechanistic rationale in Figure 1. As a matter of fact, all the camptothecin analogues including their in vivo metabolic products possess the structure feature required to be discriminated by the mechanistic pathway in Figure 1.11-13,34,35 Therefore, the two forms of each camptothecin analogue and metabolic product can be differentiated by 62 Da loss in (-)ESI-MS/MS. As to the metabolic products, other chemical alterations occur besides hydrolysis of the lactone ring.34,35 At this point, our method can be especially useful to differentiate the two forms because it uses this 62 Da neutral loss in MS/MS data, which is independent of the m/z for the precursor ion. For example, if MS/MS spectrum of one metabolite demonstrates the 62 Da loss in (-)ESI-MS/MS, the lactone ring in this metabolite must have been hydrolyzed; on the contrary, if the 62 Da is not available, then it indicates that this metabolite still exists in its lactone form. In this way, the lactone hydrolysis in unknown metabolites can be easily identified, and no standards are required. Example 2: Hydrolysis Products of Noscapine. The advantage of using characteristic neutral losses in MS/MS to differentiate lactone and acid forms of metabolic products can be further demonstrated in the following example. In this case, noscapine (5) is heated in basic solution overnight, and the resulting products are characterized by HPLC and mass spectrometry. Figure 8a is the HPLC chromatogram. Two major peaks are present, and the eluents of both peaks are subjected to mass spectrometric detection in positive ion mode. The obtained spectra are in Figure 8b-f. Figure 8e is the (+)ESI-MS spectrum of peak 2. In this spectrum, m/z 414 is the lactone form of noscapine (5). The MS/ MS spectrum of this ion is in Figure 8f. It matches the characteristic fragmentation data in Table 1 for lactone form of 5. Figure 8b contains (+)ESI-MS data of peak 1, in which two peaks can be observed. One is m/z 432, which is the same m/z as one would expect for the acid form of this compound; and another peak, m/z 414, has the same m/z as the lactone form. MS/MS is performed to obtain structural information on these species, and spectra are shown in Figure 8, panels c and d, respectively. The characteristic neutral loss of 18 Da from parent (34) Platzer, P.; Thalhammer, T.; Reznicek, G.; Hamilton, G.; Zhang, R.; Jager, W. Int. J. Oncol. 2001, 19, 1287-1293. (35) Slatter J. G.; Schaaf, L. J.; Sams, J. P.; Feenstra, K. L.; Johnson, M. G.; Bombardt, P. A.; Cathcart, K. S.; Verburg, M. T.; Pearson, L. K.; Compton, L. D.; Miller, L. L.; Baker, D. S.; Pesheck, C. V.; Lord, R. S., III. Drug Metab. Dispos. 2000, 28, 423-433.

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ion m/z 432 is consistent with the acid form of noscapine, so it can be identified as the carboxylic acid (both the MS and MS/ MS data are consistent with this assignment.) The other peak has the same m/z as the lactone form, but its MS/MS data are different. The parent ion, m/z 414, undergoes a neutral loss of 18 Da, so it cannot be the lactone. This assertion is supported by the fact that this ion, elutes together with the acid form (in peak 1) instead of the lactone form (in peak 2). Therefore m/z 414 in peak 1 must be more structurally similar to the acid. In fact, because it shows an 18 Da loss, which is the characteristic loss for carboxylic acids of this type, m/z 414 is most likely a carboxylic acid. By using the MS/MS data to deduce the fact that m/z 414 (in HPLC peak 1) is a carboxylic acid, a reasonable structure for this compound can be postulated; it is the dehydrated form of the carboxylic acid, shown in Figure 9. The process describing the formation of this isomer is also proposed in Figure 9. After the equilibrium between the lactone and carboxylic acid has been established, the acid can undergo dehydration to form a more stable, conjugated molecule, which is an isomer of the lactone. This is simply an elimination (E2) reaction, which occurs readily under basic conditions.36 In this example, two carboxylic acids were identified using MS/MS data: one was the expected hydrolysis product, and the other was an unknown hydrolysis byproduct. The characteristic neutral losses in the CID experiment were critical for assigning the chemical structures of these ions because one of them was isomeric with the original lactone; therefore, the m/z of the peak could not be used to provide any information about hydrolysis in the lactone ring. However, by checking the characteristic fragmentation pathway in MS/MS (neutral loss of 18 Da in this example), the hydrolysis of lactone ring could be identified. This is another example that demonstrates how characteristic neutral losses can be used to identify lactone hydrolysis when more than one modification is occurring at a time. CONCLUSION A method to differentiate pharmaceutical lactones from their acid forms was developed by comparing their characteristic neutral losses in MSn experiments. The method worked for all the selected 12 compounds, and pronounced differences between the two forms were obtained. Mechanistic rationales that explain (36) Carey, F. A.; Sundberg, R. J. Polar addition and elimination reactions. In Advanced Organic Chemistry Part A: Structure and Mechanisms, 3rd ed.; Plenum Press: New York, 1993; Chapter 6.

why the two forms are distinguishable were proposed; and, based on the proposed mechanisms, the structural features that are necessary to observe the characteristic neutral losses were also identified. Using this approach, it is possible to identify lactone hydrolysis in a wide variety of species, even when other structural modifications are present. This was demonstrated with two pharmaceutically relevant examples. One potential caveat to this identification is the introduction of a carboxylic acid during biotransformation, which may reduce the structural difference between lactone and its acid form. To date, the only other method available to obtain this level of structural information about lactone hydrolysis, occurring in conjunction with other modifications, is NMR spectroscopy. While NMR is the definitive method for structural analysis, the massspectrometric approach described herein is superior to NMR, when sample consumption requirements and analysis times are

limited or when sample purity cannot be readily obtained. When studying biological transformations, sample purity and sample size are always limiting considerations, so the mass spectrometric method will clearly be useful for studying lactone hydrolysis during metabolism studies. In this paper, the applicability of this method in the ion trap mass spectrometer is demonstrated, future work includes applying these studies to a triple quadrupole mass spectrometer. ACKNOWLEDGMENT The authors gratefully acknowledge the University of Kansas New Faculty Graduate Research Fund for financial support. Received for review April 27, 2005. Accepted August 9, 2005. AC0507237

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