Analyzing Trace-Level Impurities of a Pharmaceutical Intermediate Using an LCQ Fleet Ion Trap Mass Spectrometer and the Mass Frontier Software Package Chromatography and Mass Spectrometry Application Note Mark R. Kagan1, Julian Phillips2, Carrie Liu3 1 Thermo Scientific, Piscataway, New Jersey; 2 Thermo Scientific, San Jose, CA; 3 Eisai Research Institute, Andover, Massachusetts, USA Introduction With the high cost of ringing new drugs to market, the numer of candidates a pharmaceutical company can develop is limited. Most are using increasingly sophisticated screening techniques to eliminate poor candidates early in the discovery process. To e successful, properties such as the adsorption, distriution, metaolism, excretion and toxicity (ADME/Tox) of compounds, their metaolites and impurities must e assessed quickly and accurately to characterize potential lead compounds. High-throughput analysis of impurities is particularly challenging at the low sample concentrations typical in iological matrices. The wide range of possile modifications further complicates data interpretation. With inherent high sensitivity, selectivity, and sophisticated Data Dependent operational capailities, the LCQ Fleet ion trap mass spectrometer is well suited for applications involving impurities. Rapid acquisition, interpretation and analysis of structural data is crucial for maintaining a compound screening process capale of working in a cominatorial environment. Mass Frontier, Thermo s pioneering software package for interpretation and management of mass spectra, can e configured to automatically predict fragmentation pathways from virtually any proposed molecular structure. In this study, the Fragments & Mechanisms module is used to confirm the structures of three impurities associated with a key intermediate of a pharmaceutical lead. Goal Identification of the chemical structures of impurities associated with a key intermediate of a pharmaceutical drug lead.
Experimental Conditions A pharmaceutical sample from Eisai Research Institute was sumitted for analysis. All experiments were performed on an LCQ Fleet ion trap mass spectrometer operating in the APCI mode. Separation was achieved using a Surveyor LC system equipped with an AQUASIL C18 column (2 2 mm). The sample was isocratically eluted off of the column using 4% water:6% acetonitrile at a flow rate of 3 µl/min, without splitting. The following source conditions were used for the LCQ Fleet Positive ionization Heated Capillary Temp: 1 C Vaporizer Temp: 3 C APCI Discharge Current: 5 µa Sheath gas: 8 units Aux gas: 2 units Mass Frontier provides an advanced set of analytical tools designed specifically to increase the throughput of spectral interpretation. Included among its eight modules is the Fragments & Mechanisms module that enales the automatic prediction of fragmentation pathways and reaction mechanisms from user-supplied chemical structures. Based on a mathematical approach to the simulation of unimolecular ion decomposition reactions, the Fragments & Mechanisms module contains known reaction mechanisms for molecules ionized y electron impact, protonation, and chemical ionization. Discussion As part of a recent process evaluation of a key intermediate of a pharmaceutical lead, Eisai Research Institute determined that an extensive structural analysis of these intermediate s impurities was necessary efore proceeding with the next step. For proprietary reasons, only a partial structure of this compound is shown in Figure 1. The sample (2 µl of a solution containing ng/µl of the pharmaceutical intermediate and its impurities) was loaded onto the column and eluted into the mass spectrometer. The LCQ Fleet was operated in a Data Dependent MS 2 scanning mode, where oth full-scan MS and MS 2 data were acquired in a single X Figure 1: Partial structural diagram of a key pharmaceutical intermediate. Y Fragments & Mechanisms is particularly useful for: Checking the correlation etween a proposed chemical structure and its experimental mass spectrum Confirming lirary search assignments Recognizing structural differences in the spectra of closely related compounds In addition, Fragments & Mechanisms can simulate MS 2 experiments. When a user-specified compound structure is entered, a numer of theoretical secondary ion decomposition reactions are generated for comparison with experimental MS 2 data. In this study, the simulated fragmentation products of proposed structures were correlated with oserved spectra generated from Data Dependent MS 2 and used to confirm the structures of three trace-level impurities associated with a key intermediate of a pharmaceutical lead.
run without pre-specification of MS 2 precursor masses. A plot of the total ion current detected y the analysis is given in Figure 2a. A strong signal at m/z 486, corresponding to the pharmaceutical intermediate, was oserved at a retention time of 8.5 minutes. Upon detailed inspection, ackground ions (not sample-related) were discovered in the mass spectrum at m/z 391, 392 and 393. When the contriutions from these ackground ions and the pharmaceutical intermediate were sutracted from the total ion signal, three impurities at m/z 538, 488 and 556 were revealed in the resulting chromatogram, displayed in Figure 2B. Based on mass differences and isotope patterns; the three impurities were proposed to result from the addition of ClOH, H 2, and Cl 2 to the pharmaceutical intermediate s doule ond (displayed in red in Figure 1). parent drug intermediate m/z 486 dihydro impurity m/z 488 a m/z 486 Intensity Intensity hydroxychloro impurity m/z 538 dichloro impurity m/z 556 4 6 8 1 12 Time (min) 4 6 8 1 12 Time (min) Figure 2: (a) The total ion current (TIC) of the full-scan MS analysis showing elution of the drug intermediate (m/z 486) at a retention time of 8.5 minutes. () TIC with contriutions from the drug intermediate (m/z 486 and 487) and ackground ions (m/z 391, 392, and 393) sutracted out, revealing three low-level impurities at m/z 538, 488 and 556. X Y OH Cl X Y H H X Y Cl Cl a c Figure 3: Proposed chemical structure of (a) the hydroxychloro impurity () the dihydro impurity and (c) the dichloro impurity.
Figure 3 shows the proposed chemical structures of the three impurities. Figure 4 shows a comparison of the experimental isotopic patterns for the three impurities with the isotopic patterns corresponding to the proposed elemental compositions. The extracted ion chromatograms of the pharmaceutical intermediate and its three impurities are presented together in Figure 5. The signal intensity of the two chlorinated impurities was less than.1% of that of the unmodified intermediate. Sensitivity and signal-to-noise ratios were such that detection of all four of the compounds was possile in a single Data Dependent MS 2 experiment. Dynamic Exclusion, a feature of the Data Dependent acquisition software that allows the instrument to acquire a c m/z m/z Figure 4: Comparison of the experimental and theoretical isotope patterns in the protonated molecular ion regions of (a) the hydroxychloro impurity () the dihydro impurity and (c) the dichloro impurity. The experimental isotope patterns were otained from the full-scan MS data and are displayed on the left in the figure. The theoretical isotope patterns, displayed on the right in the figure, were generated y the Isotope Viewer utility in the Xcaliur software ased on the proposed elemental compositions. Figure 5: The extracted ion chromatograms of the drug intermediate and its three impurities laeled with relative peak areas.
MS 2 data for an analyte in the presence of more intense co-elutors, was enaled for this analysis. Without this feature, the MS 2 spectra of the dihydro impurity would not have een acquired, due to the presence of the more intense co-eluting unmodified intermediate. A Reject List containing the m/z s 391, 392, and 393 was also employed during this analysis to prevent these ackground ions from triggering MS 2 data acquisition. It was not necessary to specify MS 2 precursor masses prior to the analysis due to the Data Dependent nature of the acquisition. This eliminated the need to acquire a preliminary MS spectrum and then manually search it for MS 2 precursor masses, a process that is oth time-consuming and prone to failure when low-level analytes are present in the sample. In Figure 6, Mass Frontier s Spectra Manager window is used to display the experimentally acquired MS 2 spectrum of the pharmaceutical intermediate efore and after correlation with predictions made y the Fragments & Mechanisms. Specifically, the chemical structure of the pharmaceutical intermediate was sumitted to the program, which then used known unimolecular decomposition reactions to predict possile MS 2 fragment ions. These theoretically predicted MS 2 fragment ions were then matched to the experimental data. Figure 6 shows that all of the major experimental MS 2 peaks were accounted for y the program; sustantiating the utility of the Fragments & Mechanisms algorithm for predicting the MS 2 spectra of sumitted chemical structures. a 8 316 454 8 316 454 Figure 6: (a) The experimental MS 2 spectrum of the drug intermediate. () The same spectrum after correlation with theoretically generated fragment ions. Experimental fragments that were accounted for y the Mass Frontier predictive algorithm are colored in red.
Figure 7 shows the experimental MS 2 spectra of the three impurities after correlation with the fragment ions predicted y Mass Frontier s Fragments & Mechanisms module. Mass Frontier s predictions were ased on the chemical structures proposed for each of the three impurities displayed in Figure 3. As in Figure 6, the experimental MS 2 peaks that corresponded to fragment ions predicted y Mass Frontier are colored in red. The oservation that all of the major peaks in the experimental MS 2 spectra of the impurities are colored red clearly supports the validity of the proposed structures. Tale 1 summarizes the results of the correlation etween Mass Frontier s fragmentation predictions and the experimental MS 2 spectra that are used to verify the structures proposed for the impurities in Figure 3. In order to understand how this is done, it is important to recognize that except for the sustitution site (colored in red in Figures 1 and 3), the proposed structures of the drug intermediate and the three impurities are identical. Therefore, if the experimental MS 2 spectra of the four compounds each possess a fragment ion at m/z values that Mass Frontier assigns to the same mechanism of formation, the structures proposed for the three impurities are correct: The fragment ion should have the same m/z value in all four experimental MS 2 spectra if the Mass Frontier predicted mechanism of formation produces a fragment structure that does not contain the sustitution site. The fragment ion should have experimental m/z values differing y the mass of the sustituents if the Mass Frontier predicted mechanism of formation produces a fragment structure that does contain the sustitution site (i.e. if the fragment ion has a m/z value of M in the drug intermediate s experimental MS 2 spectrum then it should have m/z values of M+2, M+52, and M+7 in the experimental MS 2 spectra of the hydroxychloro, dihydro, and dichloro sustituted impurities, respectively). In Tale 1, fragment ions that have een assigned the same mechanism of formation y Mass Frontier are highlighted with the same color. Taking this into account the tale reveals that in all cases, fragment ions related y a common Mass Frontier predicted mechanism of formation either have the same experimental m/z value or have experimental m/z values that differ y the mass of the sustituents depending on whether or not the fragment structure resulting from the Mass Frontier prediction contains the proposed site of sustitution. This oservation provides the main verification for the impurity structures that were proposed. a 8 368 6 16 17 18 19 2 21 22 23 24 2 26 27 28 29 3 31 32 33 34 3 36 37 38 39 4 41 42 43 44 4 46 47 48 49 51 8 386 524 17 18 19 2 21 22 23 24 2 26 27 28 29 3 31 32 33 34 3 36 37 38 39 4 41 42 43 44 4 46 47 48 49 51 52 456 318 c 21 215 22 2 23 235 24 245 2 5 26 265 27 2 28 285 29 295 3 35 31 315 32 3 33 335 34 345 3 355 36 365 37 3 38 385 39 395 4 45 41 415 42 4 43 435 44 445 4 455 Figure 7: The experimental MS 2 spectra of (a) the hydroxychloro impurity () the dichloro impurity and (c) the dihydro impurity after correlation with theoretically generated fragment ions. Experimental fragments that were accounted for y the Mass Frontier predictive algorithm are colored in red.
Conclusions With the help of Data Dependent MS 2 scanning with Dynamic Exclusion, three impurities of a proprietary pharmaceutical intermediate were detected at or elow the.1% level and targeted for characterization. Isotopic patterns and mass differences provided the rationale for proposed structures that were sumitted to Mass Frontier for verification. The structures proposed for the impurities were confirmed y a comparison of Mass Frontier s fragmentation predictions with the experimental MS 2 spectra. The analysis supported the conclusion that all three of the impurities resulted from chemical sustitutions on a specific doule ond in the intermediate. The characterization of impurities present in amounts as low as.1% of the parent aundance is desirale from a regulatory standpoint. The LCQ Fleet ion trap mass spectrometer provides the sensitivity, selectivity, and requisite Data Dependent scanning tools for successful and compliant impurity analysis. Mass Frontier s Spectra Manager and Fragments & Mechanisms modules facilitate structural analysis y offering rapid, visual confirmation of proposed impurity structures. Compound Experimental MS 2 Fragment Mass (m/z) Does Corresponding Fragment Structure Predicted y Mass Frontier Contain Proposed Site of Sustitution? (YES/NO) Tale 1: Summary of Mass Frontier s Structural Predictions Drug Intermediate Hydroxychloro Sustituted Impurity Dichloro Sustituted Impurity Dihydro Sustituted Impurity 8 No 316 Yes No No 454 Yes 8 No 368 Yes No No 6 Yes 8 No 386 Yes No No 524 Yes 318 Yes 456 Yes Note: The dihydro impurity eluted on the tail of the pharmaceutical intermediate s chromatographic peak, which also contained a significant contriution from m/z 488 in its mass spectrum. Since m/z 488 is the precursor mass used to otain MS 2 spectra for the dihydro impurity, it was necessary to sutract out the contriution of the pharmaceutical intermediate from the MS 2 spectrum of the dihydro impurity. This removed the fragment ions that were common to oth. For this reason, the aove tale and Figure 7 only contains fragment ions for the dihydro impurity that differ in mass from those of the pharmaceutical intermediate. See Figures 1 and 3 for details.