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;

Thermo Scientific, San Jose, CA;

Eisai Research Institute, Andover, Massachusetts, USA

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 (ADME/Tox) 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 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 capable of working in a combinatorial
environment. Mass Frontier, Thermos 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

Discussion

A pharmaceutical sample from Eisai Research Institute

was submitted 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 (250 2 mm).
The sample was isocratically eluted off of the column
using 40% water:60% acetonitrile at a flow rate of
300 L/min, without splitting.

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 Fleet was operated
in a Data Dependent MS2 scanning mode, where both
full-scan MS and MS2 data were acquired in a single

The following source conditions were used for

the LCQ Fleet
Positive ionization
Heated Capillary Temp: 175 C
Vaporizer Temp: 325 C
APCI Discharge Current: 5 A
Sheath gas: 80 units

R1

Aux gas: 20 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
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.

R4

R2
R3

Figure 1: Partial structural diagram of a key pharmaceutical

intermediate.

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).

parent drug
intermediate
m/z 486

Intensity

Intensity

m/z 486

dihydro impurity
m/z 488

10

12

dichloro
impurity
m/z 556

hydroxychloro
impurity
m/z 538

Time (min)

10

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. (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.

R4

R2
R3

Cl

OH
R1

Cl

R1

R4

R2
R3

R1

R4

R2
R3

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

Cl

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.
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

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 (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.

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 low-level analytes are present in the sample.

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 experimental 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.

100

428
316
75

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25

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 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, 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 hydroxychloro, dihydro, 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.

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210 215 220 225 230 235 240 245 250 255 260 265 270 275 280 285 290 295 300 305 310 315 320 325 330 335 340 345 350 355 360 365 370 375 380 385 390 395 400 405 410 415 420 425 430 435 440 445 450 455

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.

Conclusions
MS2 scanning

With the help of Data Dependent

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.

Experimental
MS2 Fragment
Compound Mass (m/z )

Does Corresponding
Fragment Structure
Predicted by Mass
Frontier Contain
Proposed Site of
Substitution?
(YES /NO)

Drug Intermediate

258
316
396
428
454

No
Yes
No
No
Yes

Hydroxychloro

258
368
396
428
506

No
Yes
No
No
Yes

258
386
396
428
524

No
Yes
No
No
Yes

318
456

Yes
Yes

Substituted Impurity

Dichloro
Substituted Impurity

Dihydro
Substituted Impurity

Note: The dihydro impurity eluted on the tail of the pharmaceutical

intermediates chromatographic peak, which also contained a significant contribution from m/z 488 in its mass spectrum. Since m/z 488
is the precursor mass used to obtain MS2 spectra for the dihydro
impurity, it was necessary to subtract out the contribution of the
pharmaceutical intermediate from the MS2 spectrum of the dihydro
impurity. This removed the fragment ions that were common to both.
For this reason, the above table 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.

The characterization of impurities present in amounts

as low as 0.1% of the parent abundance is desirable
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 Frontiers Spectra Manager and Fragments &
Mechanisms modules facilitate structural analysis
by offering rapid, visual confirmation of proposed
impurity structures.

Table 1: Summary of
Mass Frontiers
Structural Predictions