Identification of an Adduct Impurity of an Active Pharmaceutical Ingredient and a Leachable in an Ophthalmic Drug Product Using LC-QTOF

Article abstract of DOI:10.1016/j.xphs.2019.06.009

Impurity investigations are important in pharmaceutical development to ensure drug purity and safety for the patient. The impurities typically found in drug products are degradants or reaction products of the active pharmaceutical ingredient (API) or leachable compounds from the container closure system. However, secondary reactions may also occur between API degradants, excipient impurities, residual solvents, and leachables to form adduct impurities. We hereby report an adduct-forming interaction of API (moxifloxacin) with a leachable compound (ethylene glycol monoformate) in moxifloxacin ophthalmic solution. The leachable compound originated from a low-density polyethylene bottle used in the packaging of drug products. The adduct impurity was tentatively identified as 1-cyclopropyl-6-fluoro-7-(1-(2-(formyloxy)ethyl) octahydro-6H-pyrrolo[3,4-b]pyridin-6-yl)-8-methoxy-4-oxo-1,4-dihydroquinoline-3-carboxylic acid (C₂₄H₂₈FN₃O₆, MW = 473.19621) using accurate mass LC-QTOF analysis. The mass accuracy error between the theoretical mass and the experimental mass of an impurity was found to be 0.2 ppm. An MS/MS analysis was utilized to provide mass spectrometry fragments to support verification of the proposed structure.

Full text of DOI:10.1016/j.xphs.2019.06.009

Journal of Pharmaceutical Sciences xxx (2019) 1-7
Contents lists available at ScienceDirect
Journal of Pharmaceutical Sciences
journal homepage: www.jpharmsci.org
Drug DiscoveryeDevelopment Interface
Identification of an Adduct Impurity of an Active Pharmaceutical
Ingredient and a Leachable in an Ophthalmic Drug Product Using
LC-QTOF
,
Ramarao Gollapalli 1 *, Gagandeep Singh 1, Alejandro Blinder 1, Jeremiah Brittin 2,
Arijit Sengupta 2, Bikash Mondal 1, Milan Patel 1, Biswajit Pati 1, James Lee 1,
Amit Ghode 1, Mahesh Kote 1
1 Research and Development, Akorn Pharmaceuticals, 50 Lakeview Parkway, Suite 112, Vernon Hills, Illinois 60061
2 Mund-Lagowski Department of Chemistry and Biochemistry, Bradley University, Peoria, Illinois 61625
a r t i c l e i n f o
a b s t r a c t
Article history:
Impurity investigations are important in pharmaceutical development to ensure drug purity and safety
for the patient. The impurities typically found in drug products are degradants or reaction products of the
active pharmaceutical ingredient (API) or leachable compounds from the container closure system.
However, secondary reactions may also occur between API degradants, excipient impurities, residual
solvents, and leachables to form adduct impurities. We hereby report an adduct-forming interaction of
Received 4 December 2018
Revised 21 May 2019
Accepted 7 June 2019
API (moxifloxacin) with
a leachable compound (ethylene glycol monoformate) in moxifloxacin
Keywords:
active pharmaceutical ingredient
extractables
leachables
adduct
low-density polyethylene
LC-QTOF
ophthalmic solution. The leachable compound originated from a low-density polyethylene bottle used
in the packaging of drug products. The adduct impurity was tentatively identified as
1-cyclopropyl-6-fluoro-7-(1-(2-(formyloxy)ethyl) octahydro-6H-pyrrolo[3,4-b]pyridin-6-yl)-8-methoxy-
4-oxo-1,4-dihydroquinoline-3-carboxylic acid (C24H28FN3O6, MW ¼ 473.19621) using accurate mass LC-
QTOF analysis. The mass accuracy error between the theoretical mass and the experimental mass of an
impurity was found to be 0.2 ppm. An MS/MS analysis was utilized to provide mass spectrometry
fragments to support verification of the proposed structure.
© 2019 American Pharmacists Association®. Published by Elsevier Inc. All rights reserved.
Introduction
drug product.2,3 The International Conference on Harmonization
Q3B provides guidance on the control of impurities in drug prod-
The purity of a pharmaceutical drug product is of paramount
importance to ensure drug safety from the standpoint of patient
exposure to impurities. Although precautions are always imple-
mented to optimize excipient selection, formulation process,
manufacturing process, and the appropriate packaging selection,
impurities are still likely to be encountered. It is mainly due to
degradation pathways of active pharmaceutical ingredients (API)1
and due to leachable compounds originating from the container
closure system (CCS) or the manufacturing contact surfaces of the
ucts.4 The United States Pharmacopeia (USP) recently added
chapter <1664> to provide guidance on the testing of leachable
compounds. It is important that highly selective and sensitive
analytical methods are in place for the impurity investigations
during the product development phase.5,6 If any impurity is iden-
tified at a level representing toxicological risk, it should be
adequately quantified and evaluated for safety assessments.7,8 For
example, upon structural elucidation, an impurity can be evaluated
utilizing approaches such as Quantitative Structure Activity Rela-
tionship analysis.7,9 Furthermore, based on the identification of the
impurity and its source, attempts could be made to control or avoid
its formation.10
Conflict of interest: The authors declare no competing financial interest.
The authors Ramarao Gollapalli and Gagandeep Singh contributed equally.
This article contains supplementary material available from the authors by request
or via the Internet at https://doi.org/10.1016/j.xphs.2019.06.009.
The impurities typically found in drug products are degradants
or reaction products of the API, or leachable compounds from
packaging components.1,11 However, it is important to have an in-
depth understanding of the degradation pathways of API in the
product. Furthermore, secondary reactions may also occur between
* Correspondence to: Ramarao Gollapalli (Telephone: þ1-216-375-4376).
E-mail address: ramarao.gollapalli@akorn.com (R. Gollapalli).
https://doi.org/10.1016/j.xphs.2019.06.009
0022-3549/© 2019 American Pharmacists Association®. Published by Elsevier Inc. All rights reserved.

2
R. Gollapalli et al. / Journal of Pharmaceutical Sciences xxx (2019) 1-7
API degradants, excipient impurities, residual solvents to form
adduct impurities.12 In addition to the secondary reactions as just
mentioned, the interaction of API with leachable impurities is
another possibility which cannot be ruled out. In such cases, the
identification process becomes more complex and requires the
utilization of advanced analytical techniques. Various types of
drug-excipient interactions in pharmaceutical drug development
are well documented.13-17 There are also several reports on re-
actions of API with its degradation products.12,18,19 However, to the
best of our knowledge, reports of adduct-forming interactions of
API with leachables are limited.
Standard and Sample Preparation for GC-HS-MS Analysis
A standard was prepared by transferring 115 L of toluene
m
(density 0.87 g/mL) into a 100 mL volumetric flask containing
approximately 20 mL of DMSO. Then the flask was diluted to vol-
ume with DMSO and mixed well. Pipetted 1.0 mL of this solution
into a 100 mL volumetric flask and diluted to volume with DMSO
and mixed well (concentration of toluene ~ l0 ppm). Pipetted 1.0
mL of this solution into a 20 mL headspace vial and crimped to seal.
For test articles (bottles, caps, and tips) approximately 1 g were
accurately weighed and placed into individual headspace vials and
capped for analysis.
One such phenomenon was observed for an ophthalmic product
where API (moxifloxacin) interacted with a low-density poly-
ethylene (LDPE) container over time to yield an unknown impurity.
We recently reported on an identification of Irganox-related leach-
able impurities and on migration of diethyl phthalate in ophthalmic
drug products20,21 and hereby present a case study demonstrating
the identification of an impurity formed from a reaction of an API
molecule (moxifloxacin) with a leachable compound (ethylene gly-
col monoformate originated from LDPE). This impurity was detected
at ~0.3% (w/w) in moxifloxacin ophthalmic solution. The maximum
daily dosage of the drug product is 3 drops per day and the identi-
fication threshold for the unknown impurity is 0.1% as per USP
monograph. The impurity was tentatively identified as
1-cyclopropyl-6-fluoro-7-(1-(2-(formyloxy)ethyl)octahydro-6H-pyr-
rolo[3,4-b]pyridin-6-yl)-8-methoxy-4-oxo-1,4-dihydroquinoline-
3-carboxylic acid (C24H28FN3O6, MW ¼ 473.19621) using accurate
mass LC-QTOF analysis. The mass accuracy error between the
theoretical mass and the experimental mass of an impurity was
found to be 0.2 ppm. An MS/MS analysis was utilized to provide
mass spectrometry fragments to support verification of the pro-
posed structure. As the impurity contains possibly alerting alde-
hyde group in its structure, it was important to identify and
control the impurity in the drug product to ensure drug safety.
Extractable Study on Primary Packaging Components (LDPE Bottle,
LDPE Cap and Tip)
The extractable study was performed using 3 types of extraction
solvents: (1) buffer pH 3 (0.1 M sodium phosphate); (2) buffer pH 9
(0.1 M sodium bicarbonate); and (3) ethanol and water (40:60 v/v).
Approximately, 5 g of test articles were weighed into separate glass
containers containing 200 mL of each extraction solvent. After
30 min of sonication, the units were placed in an oven maintained
at a temperature of 50ꢁC for 4 days. Control samples were prepared
similarly by placing the same volume of extraction solvents in glass
bottles without test articles. After 4 days, the samples were taken
out and injected as is for LC-MS analysis. For GC-MS analysis, each
extraction solvent was subjected to solvent extraction using
dichloromethane. The organic layers for each test article were
combined and the resulting solution was concentrated to ~1 mL by
blowing nitrogen over the top of the solution, transferred to a GC
vial and analyzed using GC-MS method.
GC-MS Analysis
The GC-MS analysis was performed on an Agilent Gas Chro-
matography system 7890A (Agilent Technologies, Santa Clara, CA),
equipped with a Mass Spectrometry triple-axis 5975C detector.
Agilent Mass Hunter software was used for data collection and
instrument control. The chromatographic separation was achieved
Materials and Methods
Chemicals and Materials
using an Agilent HP-5MS column, 30 m ꢂ 0.25 mm, 0.25
mm
(Agilent, Serial # USN723024H). Helium was used as a carrier gas at
a flow rate of 2.0 mL/min. A splitless injector was used and the
injector temperature was set to 300ꢁC. The MS transfer line was
maintained at 250ꢁC and the ionization mode was electron impact
at 70 eV. The initial oven temperature was 40ꢁC, maintained for
5 min, followed by a temperature ramp to 280ꢁC @ 10ꢁC/min,
maintained for 3 min, and another temperature ramp to 320ꢁC
@15ꢁC/min, maintained for 5 min.
High pressure liquid chromatography grade methanol (MeOH)
and acetonitrile (ACN) was obtained from Fisher Scientific, Fairlawn,
NJ. Formic acid (purity, ꢀ98%), dimethyl sulfoxide (DMSO) (purity,
ꢀ99%) and toluene (purity, ꢀ99%) were purchased from Sigma-
Aldrich, St Louis, MO. Moxifloxacin HCl (anhydrous) was obtained
from MSN Pharmachem Pvt. Ltd. Andhra Pradesh, India. The ultra-
pure water used was purified by a MilliQ water system (Millipore,
France). ACS grade ammonium acetate, formic acid, sodium phos-
phate monobasic, and sodium bicarbonate were obtained from
Fisher Scientific. Ethanol (purity, ꢀ99%) and dichloromethane
(ꢀ99.9% grade) were purchased from Sigma-Aldrich.
LC-MS Analysis
The LC-MS accurate mass analysis was performed on a liquid
chromatographic system model 1290 (Agilent Technologies, Palo
Alto, CA) coupled to a 6530 accurate-mass QTOF mass spectrometer
equipped with a dual jet stream electrospray ionization (ESI)
interface. Agilent Mass Hunter software was used for data collec-
tion and instrument control.
Sample Preparation for LC-MS Analysis
Moxifloxacin ophthalmic solution (in house), 0.5% Lot#
PD13018, CRT, 24M, was used as a drug product sample. Each mL of
moxifloxacin ophthalmic solution contains 5.45 mg moxifloxacin
hydrochloride, equivalent to 5 mg moxifloxacin base. The other
ingredients include boric acid, sodium chloride, and purified water.
The pH of the solution is adjusted to approximately 6.8 using hy-
drochloric acid/sodium hydroxide. 0.5 mL of the drug product
sample was diluted with 0.5 mL of water and mixed well for LC-MS
analysis.
LC-QTOF Method
The chromatographic separation was achieved using a gradient
elution (flow rate: 0.9 mL/min) on a Zorbax SB-Phenyl column
150 ꢂ 3.0 mm, 3.5
mm (Agilent, Part # 863954-312). 0.1% formic
acid was used as solvent A and methanol as solvent B. The
following elution gradient was applied, 0-4.7 min: %B ¼ 31, 4.7-
5.1 min: %B ¼ 31-40, 5.1-10.3: % B ¼ 40, 10.3-10.4: %B ¼ 40-31,

R. Gollapalli et al. / Journal of Pharmaceutical Sciences xxx (2019) 1-7
3
Figure 1. Typical LC-MS total ion chromatogram (expanded version) of moxifloxacin ophthalmic solution sample.
10.4-14.0: %B ¼ 31. The UV data were collected at 237 nm. The
injection volume of the method was 1 L and the analytical col-
Spectrometry triple-axis detector 5975C. Agilent ChemStation
software was used for data collection and instrument control. The
chromatographic separation was achieved using an Agilent HP-5MS
m
umn was maintained at 45ꢁC. MS analysis was performed in
positive mode using an ESI interface. The gas temperature was set
to 300ꢁC and sheath gas temperature to 250ꢁC at a flow rate of 11
L/min. The nebulizer pressure was set to 45 psi and the capillary
voltage to 3500V. In addition, the fragmentor voltage was set to
75V, skimmer to 65V and Oct 1 RF voltage to 750V.
column, 30 m ꢂ 0.25 mm, 0.25
mm (Agilent, Serial # USN723024H).
Helium was used as a carrier gas at a flow rate of 2.0 mL/min. A split
injector (split ratio: 5:1) was used and the injector temperature was
set to 200ꢁC. The MS transfer line was maintained at 250ꢁC and the
ionization mode was electron impact at 70 eV. The initial oven
temperature was 40ꢁC, maintained for 3 min, followed by a tem-
perature ramp to 250ꢁC @ 10ꢁC/min. The solvent delay for 2 min
was used. For the standard injection, the MS state was turned off
after 4.5 min to avoid DMSO (diluent) entering the MS source.
MS/MS Method
Same conditions were used as described in LC-QTOF method. In
addition, the collision energy of 35V and the cell accelerator voltage
of 4V were applied during the experiment to obtain MS/MS data for
the precursor ion of m/z 274.2034.
Synthesis of Ethylene Glycol Monoformate
GC-HS-MS Analysis
Ethylene glycol (5.01 g) was treated with formic acid (3.34 g)
and the resulting mixture was stirred at 55ꢁC for 48 h. The crude
product was purified by column chromatography (silica gel) using
ethylacetate/hexane mixture as the eluent (70:30). The solvent was
GC-MS analysis was performed on an Agilent Gas Chromatog-
raphy system 7890A (Agilent Technologies), equipped with a Mass
Figure 2. Mass spectrum of moxifloxacin (top) and the impurity RRT 1.2 (bottom).

4
R. Gollapalli et al. / Journal of Pharmaceutical Sciences xxx (2019) 1-7
removed under reduced pressure to obtain a colorless liquid. Yield:
35%. Purity: >95%. 1H NMR (400 MHz, CDCl3, ppm): 8.08 (1H, s),
4.26 (2H, t), 3.82 (2H, t), 2.44 (1H, br).
retention time of ~28.6 min and an unknown impurity (RRT 1.2) at a
retention time of ~35.6 min (see Supportive Fig. S1). This impurity
was observed to increase over a period of time and was detected at
~0.3% in stability samples stored under controlled room tempera-
ture (CRT) conditions for 24 months. Initially, the UV spectrum of
this impurity (RRT 1.2) was compared with the moxifloxacin API in
the ophthalmic drug product. The UV data exhibited an identical
profile (see Supportive Fig. S2), which suggested that the impurity
at RRT 1.2 is moxifloxacin-related and its core structure was intact.
Forced degradation (stress) studies were conducted on API and
drug product without being in contact with its CCS to understand if
the impurity at RRT 1.2 is a possible degradant of moxifloxacin. This
impurity was not formed under any stress condition (acidic-basic
hydrolysis, oxidation, and photodegradation), which indicated that
it might not be a degradant of the moxifloxacin molecule. A thor-
ough literature search was conducted and this impurity was not
referenced anywhere. Therefore, a systematic study was designed
to identify this impurity and its source.
Spike Studies
Approximately, 20 mg of moxifloxacin API was weighed and
transferred into a 20 mL scintillation vial and 4 mL of deionized
water was added. The resulting solution was mixed well and 2 mL
of this solution was transferred into 2 separate scintillation vials.
One of these vials was labeled as control and in other vial con-
taining moxifloxacin solution, 0.5 mL of ethylene glycol mono-
formate was spiked and the resulting solution was mixed well. The
control and the spiked sample were placed into an oven maintained
at 50ꢁC for analysis at different time points using LC-MS analysis.
Results and Discussion
Moxifloxacin
(1-cyclopropyl-6-fluoro-8-methoxy-7-(octahy-
To have a better insight into the molecular structure of the
impurity, an LC-MS method was developed (see experimental
section for method details). This method was developed by utiliz-
ing accurate mass LC-QTOF, and was able to resolve the impurity of
interest from the API and matrix-related peaks in the drug product
yielding important structural information. Figure 1 depicts the LC-
MS total ion chromatogram of the drug product exhibiting API,
matrix, and impurity peaks. The peak at RT ~7 min corresponds to
moxifloxacin and the peak at RT ~10 min is the impurity peak of
dro-6H-pyrrolo[3,4-b]pyridin-6-yl)-4-oxo-1,4-dihydroquinoline-3-
carboxylic acid) is a fourth-generation quinolone that has been
significantly studied over the past decade and its ophthalmic so-
lution is widely used to treat bacterial conjunctivitis or other bac-
terial infections of the eyes.22 An unknown impurity was observed
during the stability testing of in-house moxifloxacin ophthalmic
solution at RRT 1.2 using high pressure liquid chromatography
analysis (USP monograph, USP40eNF35). The API eluted at a
a
b
Scheme 1. (a) The proposed fragmentation of the molecular ion m/z 474.20341(impurity RRT 1.2). (b) Proposed structure and structural formula of the impurity at RRT 1.2.

R. Gollapalli et al. / Journal of Pharmaceutical Sciences xxx (2019) 1-7
5
Figure 3. Overlay of LC-MS extracted ion chromatograms (EICs at m/z ¼ 474) of spiked sample (from top to bottom) at ~ 1 week, ~ 2 weeks and ~1 month.
interest (RRT 1.2). The MS spectra of both peaks are illustrated in
Figure 2. The impurity at RRT 1.2 yielded a molecular ion of m/z
474.2034 [M þ H]þ, whereas moxifloxacin (API) yielded a molecular
ion peak of m/z 402.1818 [M þ H]þ in positive mode using ESI. In the
mass spectrum of moxifloxacin, m/z 402.1818 represents molecular
ion [M þ H]þ, and m/z 803.3570 represents the dimer [2M þ H]þ.
Similarly, in the mass spectrum of the impurity, m/z 474.2034
represents the impurity molecular ion [M þ H]þ and m/z 947.3986
represents the dimer [2M þ H]þ. The molecular formula database
search in the Agilent Mass Hunter software suggested the tentative
structural formula as C24H28FN3O6 (exact mass ¼ 473.19621 Da).
The theoretical mass (M þ H) for the molecular ion was calculated
as 474.2035 and the measured mass obtained from Q-TOF analysis
was 474.2034. The mass accuracy error between the theoretical
mass and the experimental mass of an impurity was found to be 0.2
ppm. Based on this data, the experimental molecular formula was
found to be consistent with the theoretical one with greater
accuracy.
structure.6 Therefore, we continued our study to determine the
actual source of the impurity RRT 1.2 and to elucidate its tentative
structure. The mass unit difference of 72 between the moxifloxacin
and the impurity molecule along with the fact they exhibited
identical UV spectra suggested that this impurity could be an
adduct of moxifloxacin molecule and another moiety present in its
surrounding. Based on this background information, the plausible
interactions of moxifloxacin molecule were assessed. Initially, the
excipients and the known degradants of moxifloxacin were
screened for potential sources of this impurity with no conclusive
outcome. To evaluate the individual components of the CCS (bot-
tles, caps, and tips), an extractable study was performed following
PQRI guidelines3 for ophthalmic drug products. The details of this
study are provided in the experimental section. Through systematic
extraction of these container closure components and subsequent
MS analysis, extracted compound ethylene glycol monoformate
(C3H6O3, MW ¼ 90.078 Da) originating from a LDPE bottle, pre-
sented itself as a potential source of impurity. A typical chro-
matogram from a GC-HS-MS analysis of the LDPE bottle exhibiting
ethylene glycol monoformate peak at RT 3.4 min is presented in
Proactive management and control of foreign impurities is most
effectively accomplished by knowing its source and possible

6
R. Gollapalli et al. / Journal of Pharmaceutical Sciences xxx (2019) 1-7
Figure 4. MS-MS spectrum of impurity RRT 1.2 present in the drug product.
Supportive Figure S3a. The electron impact GC-MS spectrum of
ethylene glycol monoformate peak is illustrated in Supportive
Figure S3b which matched very well (match factor >80%) with its
reference mass spectrum in the NIST library database. It was hy-
pothesized that the impurity RRT 1.2 (C24H28FN3O6, MW ¼
473.19621 Da) could be originating from an interaction of moxi-
floxacin and ethylene glycol monoformate. The proposed structure
of the adduct impurity (RRT 1.2) is depicted in Scheme 1a.
As the impurity RRT 1.2 is a new compound, it is not commer-
cially available and the route of synthesis is not available in the
literature. An MS/MS analysis was utilized to provide mass spec-
trometry fragments to support further verification of the proposed
structure. LC/MS/MS is considered a very powerful tool to obtain
ion fragmentation patterns for unknown compounds. The loss of
neutral molecules and fragmentation ions in the MS/MS processes
are generally considered very informative for the structure eluci-
dation of targeted molecules. This can greatly facilitate the under-
standing of ion fragmentation pathways for an unknown species,
from which the identity of unknown compounds can be proposed.
The collision energy of 35 V was applied in the MS/MS analysis to
dissociate the impurity peak into smaller fragments. Figure 4 shows
the MS/MS spectrum of the impurity RRT 1.2 present in the drug
product yielding important information about its fragmentation.
The most abundant fragments of m/z 456.1932 and m/z 168.1020 in
the MS/MS spectrum represent a dehydrated impurity molecule
(m/z 474.2034-H2O) and the piperidine moiety with an ethylene
monoformate chain, respectively. The proposed mass spectro-
metric fragmentation pathway is demonstrated in Scheme 1b along
with the possible structures of the major fragments.23 It is to be
noted that the fragmentation pattern observed for this impurity
aligns with that of moxifloxacin reported in the literature23 except
the fragments (m/z 168 and m/z 456) which are unique to the
impurity structure. The accurate masses and elemental composi-
tions of the major product ions are listed in Table 1. Based on this
MS/MS data, the molecular formula of the impurity RRT 1.2 was
established and the tentative structure is shown in Scheme 1a.
Based on our results, it was determined that impurity RRT 1.2 is
potentially formed because of the interaction of moxifloxacin with
ethylene glycol monoformate (originated from LDPE) during the
storage of the drug product. The optimal conditions to form this
impurity are unknown. It is understood that LC/MS alone some-
times cannot reveal or confirm the final structures as required.
However, relating the precursor information with the fragments
observed in the MS/MS analysis, structural elucidation of impurity
RRT 1.2 was proposed. The structure of the impurity contains
possibly alerting aldehyde group; hence it is important to control
the impurity in the drug product.
To prove our hypothesis, ethylene glycol monoformate was
synthesized according to the procedure discussed in Section
Synthesis of Ethylene Glycol Monoformate. The 1H NMR spectrum
of ethylene glycol monoformate has been presented in the
Supportive Figure S4. The aldehyde proton exhibited chemical shift
of 8.08 ppm and ethylene protons appeared at 3.82 ppm and 4.26
ppm. The aqueous solution of moxifloxacin (5 mg/mL) was pre-
pared and divided into 2 aliquots of equal quantities, one spiked
with ~0.5 mL ethylene glycol monoformate and the other unspiked.
Both spiked and unspiked (control) samples were placed in an oven
maintained at 50ꢁC and analyzed using LC-MS at different time
points that is 1 week, 2 weeks, and 1 month. The sample solution
containing ethylene glycol monoformate revealed the impurity RRT
1.2 peak, whereas the control sample did not. Moreover, the
amount of impurity appeared to grow with time. Owing to the low
amounts of the impurity observed, the impurity peak (m/z ¼ 474)
was extracted from the LC-MS total ion chromatogram and pre-
sented as overlay at different time points. The overlay of extracted
ion chromatograms of spiked sample is presented in Figure 3 which
depicts the growth of impurity with time. The overlay of extracted
ion chromatograms of control sample at different time intervals is
shown in Supportive Figure S5. The presence of impurity peak in
the spiked sample with ethylene glycol monoformate was
confirmed by extracting its mass spectrum. In addition, the reten-
tion time of the impurity generated by this experiment is exactly
matching with the impurity seen in moxifloxacin HCl ophthalmic
solution stability samples. This further strengthened the proposed
source of the impurity formation.
Table 1
Proposed Fragments of the Impurity Molecule
m/z
Fragmentation
474.2034
456.1932
413.1746
298.1114
260.0597
245.0718
168.1020
(M þ H)
Conclusion
(M þ H-H2O)
M þ H-HF-C2H3N
During pharmaceutical drug development, identification of im-
purities and assessment of their toxicityare required by the FDA. The
common approach when an unknown impurity needs to be iden-
tified is to look at the possible degradation pathways of the API
Fragment shown in Scheme 1b
M þ H-HF-C2H3N-C8H12NO2
M þ H-HF-C2H3N-C8H12NO2-CH3
Fragment shown in Scheme 1b

R. Gollapalli et al. / Journal of Pharmaceutical Sciences xxx (2019) 1-7
7
5. ICH Harmonised Tripartite Guideline. Pharmaceutical Development, Q8(R2).
2005.
followed by the possible reactions of the degradants with the drug
product excipients. However, in this study, we demonstrated that
leachables can result in impurities formation as well. Not only
leachables can be encountered as possible impurities, but also as
possible adducts with API or related compounds. Identification of
leachables and degradation products of API in a drug product is a
challenging task in itself. Identification of entirely new compounds
formed through cross-reactivity between impurities, leachables and
API, presents yet another layerof complexity. The impurityat RRT 1.2
in moxifloxacin ophthalmic solution was tentatively identified by
accurate mass LC-QTOF. The chemical name and the formula were
proposed as 1-cyclopropyl-6-fluoro-7-(1-(2-(formyloxy)ethyl)
octahydro-6H-pyrrolo[3,4-b]pyridin-6-yl)-8-methoxy-4-oxo-1,4-
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14. Chadha R, Bhandari S. Drugeexcipient compatibility screeningdrole of ther-
moanalytical and spectroscopic techniques. J Pharm Biomed Anal. 2014;87:82-
97.
dihydroquinoline-3-carboxylic acid and
C
24H28FN3O6 (MW
¼
473.19621), respectively. The origin of this impurity has been iden-
tified as the interaction of moxifloxacin with ethylene glycol mon-
oformate. This impurity was observed at ~0.3% in moxifloxacin
stability samples and contains aldehyde group in its structure which
is alerting for safety perspective. Therefore, it was important to
identify and control this impurity to ensure drug safety.
15. Wu Y, Levons J, Narang AS, Raghavan K, Rao VM. Reactive impurities in ex-
cipients: profiling, identification and mitigation of drugeexcipient in-
compatibility. AAPS PharmSciTech. 2012;12(4):1248-1263.
Acknowledgments
16. Bharate SS, Bharate SB, Bajaj AN. Interactions and incompatibilities of phar-
maceutical excipients with active pharmaceutical ingredients: a comprehen-
sive review. J Excip Food Chem. 2016;3:1131.
17. Hotha KK, Roychowdhury S, Subramanian V. Drug-excipient interactions: case
studies and overview of drug degradation pathways. Am J Analyt Chem. 2016;7:
107-140.
18. Alsante KM, Ando A, Brown R, et al. The role of degradant profiling in active
pharmaceutical ingredients and drug products. Adv Drug Deliv Rev. 2007;59:
29-37.
The authors are grateful for the financial support from Akorn
Pharmaceuticals Inc. They are also thankful to Mr. Felix Gallo for his
valuable suggestions throughout our work.
Authors' contributions: The manuscript was written through
contributions of all authors. All authors have given approval to the
final version of the manuscript.
19. Wu YH. The use of liquid chromatography-mass spectrometry for the identi-
fication of drug degradation products in pharmaceutical formulations. Biomed
Chromatogr. 2000;14:384-396.
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