Applications of TiCl3 as a Diagnostic Reagent for the Detection of Nitro- and N-Oxide-Containing Compounds as Potentially Mutagenic Impurities Using Ultrahigh-Performance Liquid Chromatography Coupled with High-Resolution Mass Spectrometry

Article abstract of DOI:10.1021/acs.oprd.5b00312

The ICH has strict guidelines for limiting the presence of potentially mutagenic impurities (PMIs) in marketed drugs. Therefore, it is important to fully characterize and quantitate all possible PMIs that could arise during the process of synthesizing and developing a drug. Two important and prevalent examples of PMIs are compounds containing N-oxide and nitro functional groups. TiCl₃ derivatization is an established method for determining the presence or absence of N-oxide metabolites by reduction to the corresponding amine. In this study, we demonstrate a novel application of TiCl₃ reduction combined with high-resolution UHPLC/HRMS to analyze PMIs. The results indicate that a variety of N-oxide- and nitro-containing compounds can be readily characterized by this facile platform method. In addition, we show that this chemical derivatization method can be utilized to enhance the ionization of nitro-containing compounds for LC/MS analysis.

Full text of DOI:10.1021/acs.oprd.5b00312

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Full Paper
3
Applications of TiCl as A Diagnostic Reagent for the Detection
of Nitro and N-oxide Containing Compounds as Potentially
Mutagenic Impurities (PMIs) using Ultra-High Performance Liquid
Chromatography Coupled with High Resolution Mass Spectrometry
Rong-Sheng Yang, Adam Beard, Huaming Sheng, Li-Kang Zhang, and Roy Helmy
Org. Process Res. Dev., Just Accepted Manuscript • DOI: 10.1021/acs.oprd.5b00312 • Publication Date (Web): 03 Dec 2015
Downloaded from http://pubs.acs.org on December 4, 2015
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Applications of TiCl3 as A Diagnostic Reagent for the Detection of Nitro and
N-oxide Containing Compounds as Potentially Mutagenic Impurities (PMIs)
Using Ultra-High Performance Liquid Chromatography Coupled with High
Resolution Mass Spectrometry
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Rong-Sheng Yang, Adam Beard*, Huaming Sheng, Li-Kang Zhang*, and Roy Helmy
Department of Process and Analytical Chemistry, Merck & Co. Inc., Rahway, New Jersey
07065, United States
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ABSTRACT
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The ICH has strict guidelines for limiting the presence of potentially mutagenic impurities
(PMIs) in marketed drugs. Therefore, it is important to fully characterize and quantitate all
possible PMIs that could arise during the process of synthesizing and developing a drug. Two
important and prevalent examples of PMIs are compounds containing N-oxide and nitro
functional groups. TiCl3 derivatization is an established method for determining the presence or
absence of N-oxide metabolites by reduction to the corresponding amine. In this study we
demonstrate a novel application of TiCl3 reduction combined with high resolution
UHPLC/HRMS to analyze PMIs. The results indicate that a variety of N-oxide and nitro
containing compounds can be readily characterized by this facile platform method. In addition,
we show that this chemical derivatization method can be utilized to enhance the ionization of
nitro containing compounds for LC/MS analysis.
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Keywords: PMI, UHPLC/HRMS, N-oxide, nitro, Titanium(III) Chloride, Reduction
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INTRODUCTION
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Residual impurities resulting from manufacturing and formulation, or from degradation
of the active pharmaceutical ingredient (API), may be present in pharmaceutical products.1-6
A
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subgroup of these impurities may present a potential for carcinogenic risk and therefore pose an
additional safety concern to clinical subjects and patients7-10. Regulatory agencies have identified
potentially mutagenic impurities (PMIs) as high priority in the drug development approval
process. The ICH has recently issued a guidance document outlining how to assess and control
PMIs in pharmaceuticals (ICH M7),11 which outlines the goal of maintaining these impurities
below an acceptable threshold of toxicological concern. Hence, robust analytical techniques are
needed to detect PMIs at very low concentrations.
The use of ultra-high performance liquid chromatography coupled with high resolution
mass spectrometry (UHPLC/HRMS) is one of the most common analytical methods for the
detection and characterization of PMIs.12-13 Compounds containing nitro and N-oxide
functionalities are often present as byproducts or impurities during the drug manufacturing
process.14 These moieties are two of the key structural alerting functional groups of PMIs.15
However, some of these compounds are poorly ionized using electrospray ionization (ESI).16
Thus, there is often need for extensive UHPLC/MS method development efforts in order to
characterize these compounds.
A variety of reagents have been reported in the literature for the reduction of N-oxides,
including PCl3, PPh3, Raney Ni/H2 or Pd/C, Fe/AcOH, Zn/aq NH4Cl, NaBH4/AlCl3, NH4COOH-
Pd/C, AlI3, SnCl2/HCl, TiCl4/NaI.17 In comparison to the other treatments, Titanium (III)
chloride (TiCl3) derivatization has several advantages such as its ready availability, mild room
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temperature reaction condition, high selectivity toward oxygenated species and fast reaction
time.18 The gram scale reduction of aliphatic and aromatic N-Oxides as well as aromatic nitro
groups to the corresponding amines with TiCl3 was extensively studied by Seaton and Somei et
al.19-20
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The utility of TiCl3 was further expanded for analytical purposes as a reducing reagent
for N-oxide containing drug metabolites. The use of HPLC in the analysis of N-oxides and
sulfoxides was reported as early as 1980.21 Analytical methods combining TiCl3 reduction and
LC/MS in drug metabolism studies had been established by 1997.22 Kulanthaivel et. al. further
expanded on this metabolite application, including the selectivity of TiCl3 reduction for N-
oxidation versus S-oxidation or C-hydroxylation, with a thorough investigation published in
2004.23 However, the use of TiCl3 as a diagnostic reagent in the detection of nitro and N-oxide
containing PMIs has not been explored.
By accurate mass measurement alone, it can be unclear what functional groups give rise
to any oxygen atoms present in the predicted chemical formula of an impurity. There could be
N-oxide or nitro groups present that would be considered structural alerts for PMIs or there could
be relatively less concerning carboxylic acids or alcohols in the structure of the impurity. Before
undertaking extensive scale up, isolation, MSMS, or NMR efforts in order to provide a definitive
structure, simple treatment with TiCl3/HCl can provide quick insight into the presence or absence
of N-oxide and nitro functional groups. In this study we report the use of commercially available
TiCl3/HCl reagent as a diagnostic reagent for the detection of several types of aliphatic and
aromatic N-Oxides, quinoline N-Oxide, Quinoxaline N,N’-dioxide and nitros, as well as
compounds with a combination of N-oxide and nitro groups (Scheme 1) using UHPLC/HRMS.
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In addition, we have found this chemical derivatization method can be utilized to enhance the
ionization of nitro containing compounds in LC/MS analysis.
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Scheme 1. Representative N-oxide and nitro compounds chosen for the study
Table 1. Summary of the completion of reduction by Titanium(III) Chloride in 50% aqueous acetonitrile.
Type of
N-oxidation
Extent of
reduction
Tested Compound
By-product (wt%)
Clozapine N-Oxide (1)
aliphatic N-oxide
97%
N-Oxide (3%)
7-Bromoisoquinoline N-oxide (2)
Carbadox (3)
quinoline N-oxide
Aryl-N,N'-dioxide
94%*
94%
chloroisoquinoline (6%)
N-oxide (6%)
4-Nitro-2,3-lutidine N-oxide (4)
H-4-Nitro-Phe-Oet (5)
Aryl-NO2, Aryl-NO 83%
N-hydroxylamine (17%)
N-hydroxylamine (<1%)
N-hydroxylamine (4%)
NO2 (26%)
Aryl-NO2
>99%
96%
6-chloro-2-(4-fluorophenyl)-3-nitropyridine (6) Aryl-NO2
Benzoxazino indole core of elbasvir (7)25
Aryl-NO2
74%*
*required alternate solvent system for complete reduction.
RESULTS AND DISCUSSION
Development of a Platform Approach. Methanol, ethanol, and THF have each been reported as
18-23
suitable reaction solvents for the reduction of N-oxides by TiCl3.
In the present study, 50%
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aqueous acetonitrile was found to be a simple and nearly universal reaction solvent that is easy to
work with and more closely matches mobile phase conditions used for LC/MS analysis. The
presence of water was crucial for the reaction solvent, particularly when working with nitro
groups since an abundant proton source was required for complete reduction. Moreover, the
addition of ammonium hydroxide followed by filtration were found to be important final steps
in TiCl3/HCl treatment. Ammonium hydroxide neutralizes any excess hydrogen chloride and
converts all unreacted titanium chloride to titanium dioxide.24 The precipitated titanium dioxide
may then be removed by filtration. If these final steps were not taken, the presence of partially
oxidized titanium reagent resulted in poor chromatography and a noisy background in the MS
data. This approach was found to be universally applicable to the majority of analytes studied
with a handful of easily addressed exceptions that will be discussed.
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Reduction of N-oxides. N-oxide containing impurities are often formed as byproducts in
oxidation reactions, and as discussed are important to identify as PMIs. Three N-oxide
containing molecules were selected as model compounds to represent possible PMIs: compound
1 with an N-oxide of a tertiary aliphatic amine, compound 2 with a single aryl-N-oxide, and
compound 3 with two aryl-N-oxides (Scheme 1). Each of the compounds tested was successfully
reduced following treatment with TiCl3/HCl and the results are summarized in Table 1. The
reduction of the two aryl-N-oxides of compound 3 to the corresponding quinoxaline with 94%
completion is shown in Figure 1. A characteristic shift in retention time combined with high
resolution MS data confirmed the successful reduction of compound 3. Retention of the main
component shifted from 1.5 min for compound 3 to 1.8 min for the less polar reduced
quinoxaline (compound 3a). High resolution mass spectra confirmed the exact masses of both
compound 3 and the reduced quinoxaline product (3a) with <5 mDa error. Additionally both the
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unreacted compound 3 and the reduced quinoxaline (3a) underwent in-source fragmentation and
lost a 32 Da methoxy group. When only HCl was added to the sample, no change in either
retention or mass was observed indicating that TiCl3 is necessary for reduction. Similar analysis
was undertaken for compounds 1 and 2, the results of which are summarized in Table 1.
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Figure 1. UHPLC/UV chromatograms at 254 nm of unreacted Compound 3 (A), HCl control (B) and TiCl3/HCl
treated sample (C). High resolution mass spectra of compound 3 (D) and reduced quinoxaline compound 3a (E).
With our platform approach using 50% aqueous acetonitrile as the reaction solvent,
compound 2 showed only 40% completion of reduction. However, when the reaction solvent was
changed to 100% MeOH, >99% conversion to the reduced 7-bromoisoquinoline could be
achieved. In this case, with sufficient detection limits 40% conversion would be adequate for
compound identification, even if compound 2 was present as a low level impurity. However, a
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simple switch of the reaction solvent can quickly achieve adequate conversion for accurate
quantitation of the reduced species.
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For N-oxide containing compounds similar to compounds 1-3, simple TiCl3 treatment
could quickly determine whether or not a compound contains an N-oxide functional group that
could be a structural alert. Upon TiCl3 treatment of a suspected N-oxide containing PMI, the
retention time of the peak of interest should shift and the accurate mass of the suspect compound
would change by exactly the mass of one oxygen atom for each N-oxide group present. If these
characteristic changes in retention time and accurate mass do not occur, then it would be unlikely
that the suspect compound contains an N-oxide functional group.
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Reduction of Nitro groups. As with N-oxide containing compounds, the presence of nitro
containing PMIs is important to identify in the preparation of an API. Four compounds (4-7)
each containing an aryl-nitro group were tested, with one of these compounds (4) also containing
an aryl-N-oxide. Compounds 4-6 each showed >80% reduction with the major byproduct present
being the N-hydroxylamine resulting from incomplete reduction of the nitro group (Table 1). Of
particular note was compound 6. Reduction of compound 6 to the corresponding aniline
(compound 6a) was achieved with 96% completion with 4% of the partially reduced N-
hydroxylamine remaining (Figure 2). The retention time of the major component in each
chromatogram shifted from 2.5 min for compound 6 to 2.3 min for the reduced aniline
(compound 6a). When only HCl was added to the sample, no change in either retention or mass
was observed confirming that TiCl3 is necessary for reduction. Prior to treatment with TiCl3/HCl,
the nitro containing compound 6 ionized poorly upon MS analysis with both (+)-ESI and (-)-ESI
and would be difficult to identify by MS were it present as a low level impurity. Following
reduction to the corresponding aniline, compound 6 displayed a 50 fold increase in ion
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abundance by (+)-ESI (Figure 2) as indicated by the increase in XIC peak area from 38 for m/z
253 (compound 6) to 2932 for m/z 223 (compound 6a). The lower ion abundance for compound 6
was partly the result of the electron withdrawing nature of the aryl-nitro group which renders the
pyridine nitrogen less basic and therefore less easily protonated upon analysis by (+)-ESI. This
also contributes to the earlier retention time of the reduced aniline as it should be more basic than
its nitro containing counterpart and therefore more likely to be charged – and hence earlier
eluting – in the presence of acidic mobile phase.
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Figure 2. UHPLC/UV chromatograms at 254 nm of unreacted compound 6 (A), HCl control (B) and TiCl3/HCl
treated sample (C). MS selected-ion chromatograms for compound 6 with the target mass of m/z 253 (D, E) and
compound 6a with the target mass m/z 223 (F).
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The one compound in this study for which complete reduction of the aryl-nitro group was
not achieved with the standard approach (50% aqueous acetonitrile) was compound 7, the
benzoxazino indole core of Elbasvir.25 This was due to the poor solubility of compound 7 in our
acetonitrile/water solvent system. However, in a chloroform/methanol/water solvent system,
74% conversion to the corresponding aniline was achieved (Figure 3). Compound 7 was first
dissolved in chloroform (1 mg/mL) and diluted with methanol/water (50/50) to 0.1 mg/mL and
treated with 10 µL of TiCl3/HCl reagent. Successful reduction to the corresponding aniline was
confirmed by high resolution MS with <2 mDa error. As with Compound 6, the ion abundance
of the reduced aniline (compound 7a) by (+)-ESI increases >80 fold over the corresponding nitro
containing compound (XIC peak area increases from 127 to 10760) thus facilitating
identification and quantitation by MS.
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Figure 3. UHPLC/UV chromatogram at 254 nm for the TiCl3 treated sample (A), and high resolution mass spectra
of compound 7 (B) and the reduced aniline compound 7a (C)
For nitro containing compounds similar to compounds 4-7, TiCl3 reduction will both aid
in the confirmation of the presence of the structural alerting nitro functional group as well as
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increase the limits of detection and quantitation for LC/MS analysis for such compounds through
reduction to the corresponding aniline with increased ionization efficiency. Accurate quantitation
is of particular importance for PMIs, so the increased ionization efficiency achieved by TiCl3
treatment is of vital importance for the quantitation of low levels of nitro containing PMIs.
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CONCLUSION
We have developed a broadly applicable platform method for the characterization of nitro
and N-oxide containing PMIs through reduction with a commercially available TiCl3/HCl
reagent followed by UHPLC/HRMS analysis. We have further demonstrated that in some cases
minor changes to the reaction solvent can lead to significant improvements to the extent of
conversion to the reduced product. This simple method has proven invaluable in the
identification of PMIs since with it the presence or absence of N-oxide and nitro functional
groups that are considered structural alerts can be quickly determined. Additionally, for poorly
ionizing compounds containing aryl-nitro groups, the reduction of the nitro groups to their
corresponding anilines results in an increase in ionization efficiency with ESI-MS thus aiding in
the identification and quantitation of these potentially mutagenic impurities by UHPLC/HRMS.
EXPERIMEMTAL
Chemicals and reagents. 7-Bromoisoquinoline N-oxide was purchased from Alfa Aesar (Ward
Hill, MA). 6-chloro-2-(4-fluorophenyl)-3-nitropyridine was purchased from Fisher Scientific
(Pittsburgh, PA). H-4-Nitro-Phe-OEt.HCl was purchased from Bachem America Inc. (Torrance,
CA). Clozapine N-Oxide, 4-Nitro-2,3-lutidine N-oxide, Carbadox (Vomitoxin), and Titanium
(III) chloride (ca. 12 wt. % solution in 12 wt. % hydrochloric acid) were purchased from Sigma
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Aldrich (St. Louis, MO). All solvents used for LC/UV/MS analysis were of OPTIMA LC/MS
grade purchased from Fisher Scientific (Pittsburgh, PA).
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Sample Preparation for TiCl3 Reduction. Stock solutions of the representative N-oxide and
nitro compounds were prepared at 0.5 mM (ca. 0.1~0.2 mg/mL) in aqueous acetonitrile (50% in
water) . For a typical TiCl3 reduction experiment, an aliquot of 1000 µL of N-oxide stock
solution was treated with 5 µL of TiCl3/HCl ( 4 mol equivalent of TiCl3). After the reaction
mixture was vortexed at room temperature for 1 min, 40 µL of 5N ammonium hydroxide was
added and the sample was votexed for one additional minute to precipitate titanium (as titanium
dioxide). A 500 µL aliquot was transferred to a filter vial (Thomson SINGLE StEP™ Filter
Vials 0.45 µm PTFE, Thomson Instrument Company, Oceanside, CA) for LC/MS analysis.
Similarly, the nitro containing compounds were treated with 10 µL of TiCl3/HCl because the
reduction of a nitro group to an amine group is a 6-electron process.26 Two control samples were
also prepared by mixing an aliquot of 1000 µL stock solution with 5 µL acetonitrile and 5 µL
12% HCl, respectively.
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UHPLC/HRMS analysis. LC/MS analysis was performed on a Waters Acquity UPLC system,
consisting of a binary pump, a sample manager, a TUV detector and a Waters Synapt G1 Mass
Spectrometer (Waters, Milford, MA) under positive or negative ESI conditions. The output
signal was monitored and processed using MassLynx software designed by Waters (Milford,
MA).
The separation was carried out on a Waters HSS T3 column (2.1 x 50 mm, 1.8 µm
particle size). The mobile phase consisted of 0.1% Formic Acid in both Water (mobile phase A)
and Acetonitrile (mobile phase B). The injection volume was 2 µL. Analytes were eluted using a
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gradient method consisting of an initial hold at 5% mobile phase B for 0.5 min, followed by a
linear gradient to 99% B over 2 min, then a hold at 99% B for 0.5 min. The flow rate was 0.4
mL/min, and the column temperature was set at 40oC. UV detection was set at 254 nm.
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The eluent was introduced directly into the mass spectrometer by electrospray. Source
o
o
temperature and desolvation temperature were set at 130 C and 350 C, respectively. Nitrogen
was used as both cone gas (40 liters/h) and desolvation gas (600 liters/h), and argon was used as
collision gas. The capillary voltage was set to 3 KV. The cone voltages in a range from 10 to 20
V were applied. Leucine enkephalin was used as the lock mass (m/z of 556.2772) for accurate
mass calibration and was introduced using the LockSpray interface at 10 µL/min at a
concentration of 0.5 mg/mL in 50% aqueous acetonitrile containing 0.1% formic acid. In mass
spectrometry scanning, data were acquired in centroid mode from m/z 100 to 1000. For MSMS
fragmentation of target ions, collision energies ranging from 6 to 20 V were applied.
AUTHOR INFORMATION
Corresponding Author
*adam_beard@merck.com
*li-kang.zhang@merck.com
ACKNOWLEDGMENT
The authors would like to thank Drs. Zhihong Ge, Caroline McGregor, Christopher J. Welch and
Michael H. Kress for their support on this project.
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