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Analyzing Trace-Level Impurities of a Pharmaceutical Intermediate Using ,,, an LCQ Deca Ion Trap Mass Spectrometer and the Mass Frontier ,,, Software Package

Mark R. Kagan1, Julian Phillips2, Carrie Liu3

1Thermo Finnigan, Piscataway, NJ;
2Thermo Finnigan, San Jose, CA;
3Eisai Research Institute, Andover, MA

Chemical Structure Validation

The data presented here can be acquired using the Thermo Finnigan LCQ Series of ion trap mass spectrometers

Introduction

With the high cost of bringing new drugs to market, the number 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 be successful, properties such as the adsorption, distribution, metabolism, excretion and toxicity of compounds, their metabolites and impurities must be assessed quickly and accurately to characterize potential lead compounds.

High-throughput analysis of impurities is particularly challenging at the low sample concentrations typical in biological matrices. The wide range of possible modifications further complicates data interpretation. With inherent high sensitivity, selectivity, and sophisticated Data Dependent operational capabilities, the Thermo Finnigan LCQ Series of ion trap mass spectrometers are well suited for applications involving impurities.

Rapid acquisition, interpretation and analysis of structural data is crucial for maintaining a compound screening process capable of working in a combinatorial environment. Mass Frontier, Thermo Finnigans pioneering software package for interpretation and management of mass spectra, can be 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 submitted for analysis. All experiments were performed on an LCQ Deca ion trap mass spectrometer operating in the APCI mode. Separation was achieved using a Surveyor LC system equipped with an AQUASIL C18 column (250 x 2 mm) (Thermo Hypersil-Keystone). The sample was isocratically eluted off of the column using 40% water:60% acetonitrile at a flow rate of 300 L/min, without splitting.

The following source conditions were used for the LCQ Deca: Positive ionization Heated Capillary Temp: 175 C Vaporizer Temp: 325 C APCI Discharge Current: 5 A Sheath gas: 80 units Aux gas: 20 units

Mass Frontier Software

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 enables 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 by electron impact, protonation, and chemical ionization.

Fragments & Mechanisms is particularly useful for:

Checking the correlation between a proposed chemical structure and its experimental mass spectrum

Confirming library search assignments

Recognizing structural differences in the spectra of closely related compounds

In addition, Fragments & Mechanisms can simulate MS2 experiments. When a user-specified compound structure is entered, a number of theoretical secondary ion decomposition reactions are generated for comparison with experimental MS2 data. In this study, the simulated fragmentation products of proposed structures were correlated with observed spectra generated from Data Dependent MS2 and used to confirm the structures of three trace-level impurities associated with a key intermediate of a pharmaceutical lead.

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 intermediates impurities was necessary before proceeding with the next step. For proprietary reasons, only a partial structure of this compound is shown in Figure 1.

The sample (20 L of a solution containing 100 ng/L of the pharmaceutical intermediate and its impurities) was loaded onto the column and eluted into the mass spectrometer. The LCQ Deca was operated in a Data Dependant MS2 scanning mode, where both full-scan MS and MS2 data were acquired in a single run without pre-specification of MS2 precursor masses. A plot of the total ion current detected by the analysis is given in Figure 2a. A strong signal at m/z 486, corresponding to the pharmaceutical intermediate, was observed at a retention time of 8.5 minutes. Upon detailed inspection, background ions (not sample-related) were discovered in the mass spectrum at m/z 391, 392 and 393. When the contributions from these background ions and the pharmaceutical intermediate were subtracted 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, H2, and Cl2 to the pharmaceutical intermediates double bond (displayed in red in Figure 1). 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 0.1% of that of the unmodified intermediate.


Figure 1: Partial structural diagram of a key pharmaceutical intermediate.



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.
(b) TIC with contributions from the drug intermediate (m/z 486 and 487) and background ions (m/z 391, 392, and 393) subtracted out, revealing three low-level impurities at m/z 538, 488 and 556.



Figure 3: Proposed chemical structure of (a) the hydroxychloro impurity (b) the dihydro impurity and (c) the dichloro impurity.

Sensitivity and signal-to-noise ratios were such that detection of all four of the compounds was possible in a single Data-Dependent MS2 experiment.

Dynamic Exclusion, a feature of the Data Dependent acquisition software that allows the instrument to acquire MS2 data for an analyte in the presence of more intense co-elutors, was enabled for this analysis. Without this feature, the MS2 spectra of the dihydro impurity would not have been acquired, due to the presence of the more intense co-eluting unmodified intermediate. A Reject List containing the m/zs 391, 392, and 393 was also employed during this analysis to prevent these background ions from triggering MS2 data acquisition. It was not necessary to specify MS2 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 MS2 precursor masses, a process that is both time-consuming and prone to failure when lowlevel analytes are present in the sample.


Figure 4: Comparison of the experimental and theoretical isotope patterns in the protonated molecular ion regions of (a) the hydroxychloro impurity (b) the dihydro impurity and (c) the dichloro impurity. The experimental isotope patterns were obtained 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 by the Isotope Viewer utility in the Xcalibur software based on the proposed elemental compositions.



Figure 5: The extracted ion chromatograms of the drug intermediate and its three impurities labeled with relative peak areas.

In Figure 6, Mass Frontiers Spectra Manager window is used to display the experimentally acquired MS2 spectrum of the pharmaceutical intermediate before and after correlation with predictions made by the Fragments & Mechanisms. Specifically, the chemical structure of the pharmaceutical intermediate was submitted to the program, which then used known unimolecular decomposition reactions to predict possible MS2 fragment ions. These theoretically predicted MS2 fragment ions were then matched to the experi-mental data. Figure 6 shows that all of the major experimental MS2 peaks were accounted for by the program; substantiating the utility of the Fragments & Mechanisms algorithm for predicting the MS2 spectra of submitted chemical structures.


Figure 6: (a) The experimental MS2 spectrum of the drug intermediate. (b) The same spectrum after correlation with theoretically generated fragment ions. Experimental fragments that were accounted for by the Mass Frontier predictive algorithm are colored in red.



Figure 7: The experimental MS2 spectra of (a) the hydroxychloro impurity (b) the dichloro impurity and (c) the dihydro impurity after correlation with theoretically generated fragment ions. Experimental fragments that were accounted for by the Mass Frontier predictive algorithm are colored in red.

Figure 7 shows the experimental MS2 spectra of the three impurities after correlation with the fragment ions predicted by Mass Frontiers Fragments & Mechanisms module. Mass Frontiers predictions were based on the chemical structures proposed for each of the three impurities displayed in Figure 3. As in Figure 6, the experimental MS2 peaks that corresponded to fragment ions predicted by Mass Frontier are colored in red. The observation that all of the major peaks in the experimental MS2 spectra of the impurities are colored red clearly supports the validity of the proposed structures.

Table 1 summarizes the results of the correlation between Mass Frontiers fragmentation predictions and the experimental MS2 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 substitution 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 MS2 spectra of the four compounds each possess a fragment ion at m/z values that Mass Frontier assigns to the same mechanism of formation if the structures proposed for the three impurities are correct:

The fragment ion should have the same m/z value in all four experimental MS2 spectra if the Mass Frontier predicted mechanism of formation produces a fragment structure that does not contain the substitution site.

The fragment ion should have experimental m/z values differing by the mass of the substituents if the Mass Frontier predicted mechanism of formation produces a fragment structure that does contain the substitution site (i.e. if the fragment ion has a m/z value of M in the drug intermediates experimental MS2 spectrum then it should have m/z values of M+2, M+52, and M+70 in the experimental MS2 spectra of the dihydro, hydroxychloro, and dichloro substituted impurities, respectively).

In Table 1, fragment ions that have been assigned the same mechanism of formation by Mass Frontier are highlighted with the same color. Taking this into account the table reveals that in all cases, fragment ions related by a common Mass Frontier predicted mechanism of formation either have the same experimental m/z value or have experimental m/z values that differ by the mass of the substituents depending on whether or not the fragment structure resulting from the Mass Frontier prediction contains the proposed site of substitution. This observation provides the main verification for the impurity structures that were proposed.



Conclusions

With the help of Data Dependent MS2 scanning with Dynamic Exclusion, three impurities of a proprietary pharmaceutical intermediate were detected at or below the 0.1% level and targeted for characterization. Isotopic patterns and mass differences provided the rationale for proposed structures that were
submitted to Mass Frontier for verification.

The structures proposed for the impurities were confirmed by a comparison of Mass Frontiers fragmentation predictions with the experimental MS2 spectra. The analysis supported the conclusion that all three of the impurities resulted from chemical substitutions on a specific double bond in the intermediate.

The characterization of impurities present in amounts as low as 0.1% of the parent abundance is desirable from a regulatory standpoint. The Thermo Finnigan LCQ Deca ion trap mass spectrometer provides the sensitivity, selectivity, and requisite Data Dependent scanning tools for successful and compliant impurity analysis.
Mass Frontiers Spectra Manager and Fragments & Mechanisms modules facilitate structural analysis by offering rapid, visual confirmation of proposed impurity structures.




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