Anal Bioanal Chem

DOI 10.1007/s00216-012-6393-9

PAPER IN FOREFRONT

Isolation and characterization of degradation products of moxidectin

using LC, LTQ FT-MS, H/D exchange and NMR
Atul Awasthi & Majid Razzak & Raida Al-Kassas &
David R. Greenwood & Joanne Harvey & Sanjay Garg

Received: 13 July 2012 / Revised: 24 August 2012 / Accepted: 27 August 2012

# Springer-Verlag 2012

Abstract This study aimed to evaluate the degradation

profile and pathways, and identify unknown impurities of
moxidectin under stress conditions. During the experiments,
moxidectin samples were stressed using acid, alkali, heat
and oxidation, and chromatographic profiles were compared
with known impurities given in European Pharmacopeia
(EP) monograph. Moxidectin has shown good stability under heat, while reaction with alkali produced 2-epi and 2,3
isomers (impurities D and E in EP) by characteristic reactions of the oxahydrindene (hexahydrobenzofuran) portion
of the macrocyclic lactone. Two new, previously unreported,
Electronic supplementary material The online version of this article
(doi:10.1007/s00216-012-6393-9) contains supplementary material,
which is available to authorized users.
A. Awasthi : R. Al-Kassas : S. Garg
School of Pharmacy, The University of Auckland,
Private Bag 92019, Auckland 1142, New Zealand
M. Razzak
Ancare Scientific Ltd, Auckland,
PO Box 36240, Northcote,
Auckland 0748, New Zealand

unknown degradation products, i.e. impurity 1 and impurity

2, detected after acid hydrolysis of moxidectin (impurity 2
was also observed to a lesser extent after oxidation), were
isolated from sample matrices and identified using liquid
chromatography, NMR, high-resolution FT-ICR MS, and
hydrogen/deuterium exchange studies. FTMS analysis
showed accurate mass of molecular ion peaks for moxidectin at m/z 640.38412, impurity 1 at m/z 656.37952 and
impurity 2 at m/z 611.35684, giving rise to daughter ions
traceable up to the seventh levels of MSn experiments and
supporting the proposed structures. Both unknown impurities along with moxidectin were fully characterized by 1H,
13
C, 1D HMBC and 2D (NOESY, COSY and HSQC) NMR
experiments. The interpretation of experimental data positively identified impurity 1 as 3,4-epoxy-moxidectin and
impurity 2 as 23-keto-nemadectin. The identification of
new impurities and correlation of their chromatographic
profiles with the EP method is very useful to establish the
stability profile of moxidectin and its preparations, as well
as add value to the forthcoming moxidectin finished product
European Pharmacopeia monographs.

D. R. Greenwood
School of Biological Sciences, The University of Auckland,
Private Bag 92019, Auckland 1142, New Zealand

Keywords Moxidectin degradation product .

High-resolution FT-MS and MSn . H/D exchange . NMR .
Stress study . Macrocyclic lactone

D. R. Greenwood
New Zealand Institute for Plant & Food Research Limited,
Private Bag 92169, Auckland 1142, New Zealand

Introduction

J. Harvey
School of Chemical and Physical Sciences,
Victoria University of Wellington,
PO Box 600, Wellington, New Zealand
S. Garg (*)
School of Pharmacy and Medical Sciences,
University of South Australia,
PO Box 2471, Adelaide 5001, Australia
e-mail: sanjay.garg@unisa.edu.au

23-(O-Methyloximino)-F28249 (AC 301, 423) or moxidectin (MOX) is a potent endectocide and semi-synthetic
23-methoxime derivative of nemadectin [1]. It belongs to
the structural family of milbemycins, a sub-class of macrocyclic lactone (ML) anthelmintic, well known for their
novel mode of action against a broad range of nematode
and anthropod animal parasites and approved globally for
companion and food-producing animal health industries [1].

A. Awasthi et al.

Moxidectin is of increasing importance as a new generation

drug in this role.
The milbemycins are 16-membered macrocyclic lactones
closely related to avermectins [2]. Furthermore, these two
sub-classes are produced by fermentation and subsequent
chemical modification of a macrolide moiety and have a
similar range of activities. Moxidectins synthetic precursor,
nemadectin, is isolated from the bacterium Streptomyces
cyaneogriseus subsp. [3, 4].
As shown in Fig. 1, moxidectin contains a 16-membered
macrolactone ring as part of a pentacyclic framework. It is
similar in structure to the well-known ML ivermectin B1
and differs by the absence of the disaccharide attached at
C13, the presence of an olefin-containing chain at C25 and a
methoxime moiety at the C23 position [5].
There are a number of reports published on the highperformance liquid chromatography (HPLC) analysis of
MOX as active pharmaceutical ingredients (API), in environmental, food, milk and other encompassing matrices
using different extraction and detection methods [611]. A
comprehensive methodology review by Danaher et al. on
ML outlines details of commonly used techniques for analysis of MOX in biological matrices [12]. In general, MOX is
detected by reverse phase chromatography using an ODS
column. However, none of the published work was carried
out to determine MOX and/or its degradation products
(DPs) as API or/in finished products.
A European Pharmacopeia (EP 6.8) monograph available
for MOX provides information on assay and methods for
detecting related substance with specific limits for known
and unknown impurities [6]. However, it is known that
R2
23

H3C
16

14

R1

24

O
17

15

13

25
21

12

CH3

22

18

19

R3

20

11

H 3C

H
O

10

HO

3
8a

7
5a
5

CH 3

OH
Fig. 1 Chemical structure of moxidectin: where R1 0H, R2 0N-amide,
R3 0methyl pentenyl

compendia API methods are not very useful in providing

full details of possible DPs of finished products appearing
due to specific pH of the formulations and interactions of
excipients and vehicles with the drug molecules. On the
other hand, from a formulation stability point of view, it is
misleading to specify all compendial impurities as DPs
generated during manufacturing and storage since originally
these could be API process-related impurities and therefore
may not increase with time in finished products. Consequently, these do not require profiling during stability studies. In addition, VICH guidelines GL-39 stipulate that
controlled impurities present in new drug substance need
not be monitored or specified in the new veterinary medicinal product unless they are also DPs [13]. Therefore, it is
important to find out the origin and type of all possible
impurities before setting a specification for DPs of
moxidectin.
Hence, there was a need to evaluate the suitability of the
EP 6.8 MOX method to distinguish the DPs apart from API
process impurities using forced degradation studies. A related substance reference standard available from EP 6.8 (a
mixture of impurities from A to K) can be utilized for
identification of the known impurities. Since there is no
individual MOX formulation monograph or DP reference
standard available, the major DPs observed during investigation require isolation, purification and characterization
using HPLC, high-resolution mass spectrometry (MS) and
nuclear magnetic resonance (NMR) spectroscopy techniques. Nevertheless, several MS methods for the confirmation of MOX and related compounds in API, biological and
environmental samples have been published [11, 1416].
MOX and several related compounds/homologs were identified and characterized by MS, and their fragmentation
pathways were established by high resolution as well as
electron impact mass spectra [17]. There has been a focus
on the determination of MOX and its residues with a particular emphasis on developments of new screening technologies [18, 19]. Despite an increase in analytical papers
using LC-MS/MS for the determination of MOX in environmental, residue and food samples, there has been a
general lack of detailed published pharmaceutical reports
on MOX DPs analysis and structural characterization using
high-end approaches such as high-resolution Fourier transform ion cyclotron resonance (FT-ICR) MS accurate mass
determination, multistage mass fragmentation, mechanistic
pathway analysis, hydrogen/deuterium (H/D) exchange and
use of in silico approaches [20, 21]. In addition, the use of
such comprehensive analysis was found very useful for
unambiguous characterization of DPs during our recent
work undertaken for another ML, eprinomectin [22].
Therefore, the aim of the present work was to evaluate
the EP 6.8 HPLC method to distinguish related substances
and DPs and evaluate its ability to be a stability indicating

Characterization of degradation products of moxidectin

method. Furthermore, this study also characterizes significant DPs observed during stress studies, using LC, FT-MS,
HX-MS and NMR techniques. Structure elucidation using
comparative studies of MS data and the establishment of a
degradation pathway/mechanism of decomposition are
highlighted by the proposed determinative method.

Materials and methods

Chemicals
MOX (Zhejiang Hisun Pharmaceutical Co. Ltd., Taizhou,
China) was kindly provided by Ancare Scientific Ltd
(Auckland, New Zealand). The DPs of MOX were generated, isolated and purified in-house. HPLC grade methanol
(MeOH) and acetonitrile (ACN) were purchased from
Merck (Darmstadt, Germany). Deuterated chloroform
(CDCl3) with 0.03 %v/v TMS (D, 99.8 %) was supplied
by Cambridge Isotope Laboratories Inc. (Andover, MA,
USA). Deuterated water (98 % D) was purchased from
Sigma-Aldrich (Sydney, Australia). All other analytical
reagent grade chemicals were purchased from Ajax Fine
Chem (Auckland, New Zealand). Ultrapure water (H2O)
was obtained from a Purelab Ultra water system (ELGA
LabWater, High Wycombe, Bucks, UK). Moxidectin reference standard and system suitability mix was purchased
from European Pharmacopeia (EP).
Apparatus and equipment
HPLC
HPLC analysis of samples was performed using an UltiMate
3000 Liquid Chromatographic system (Dionex Corporation,
Idstein, Germany) equipped with an on-line degasser, gradient pump, auto-injector, thermostated column compartment and photodiode array detector (all UltiMate 3000
modules). For data processing and acquisition, Chromeleon
software version 6.8 (Dionex Corporation, Idstein, Germany) was used.

respectively, the capillary temperature was 275 C and the

tube lens voltage was set to 90 V. In-source collisioninduced dissociation (CID) was used to induce incipient
fragmentation for compounds which possessed inherent stability of the molecular ion. Values between 0 and 80 V were
used so that sufficient amounts of these fragments could
enable accurate mass data to be obtained. For MSn in the
LTQ-FT ion trap, CID using He gas was set at a value of 35
70 V collision energy for sequential fragmentation of analytes. Instrument control and data collections were handled
by Xcalibur 2.0.7 SP1 software (Thermo Fisher Scientific
Inc., San Jose, CA, USA). The system was calibrated prior
to analysis of the test samples. In silico evaluation of MS
data was carried out on HiChem Mass FrontierTM (v5.1) and
ACD/MS ManagerTM (v12).
NMR spectroscopy
The 1H, 13C, 1D and 2D experiments (HMBC, NOESY,
H-1H COSY and HSQC) were carried out on 1 mgmL1
solutions of MOX and DPs in CDCl3, on an Avance III 400
NMR spectrometer (Bruker BioSpin GmbH, Rheinstetten,
Germany) using ICON-NMR real-time software. The evaluation of data was carried out with Bruker TopSpin (v2.1)
and MestReNova (v6.2.1-7569) software.
1

Isolation and purification of degradation products

Isolation of DPs from forced degradation samples was carried out on a semi-preparative flash chromatographic system
connected to a UV monitor and an external pump (Ecom
Ltd, Prague, Czech Republic). The pure compounds were
recovered in vacuo using a rotary evaporator system.
Other equipment
The analytical samples were heat stressed at 70 C in a fully
calibrated Thermotec 2000 oven (Contherm Scientific Ltd,
Petone, New Zealand). The pH of solutions was measured
by a Cyberscan 510 pH meter (Eutech Instruments,
Singapore).

Mass spectrometry

Methods

Commercial MOX as well as isolated DPs were analysed by

positive ion electrospray ionization (ESI) on a highresolution LTQ FT-ICR mass spectrometer (Thermo Scientific, San Jose, CA, USA) with a mass detection accuracy of
2 ppm [23]. Samples were introduced into the mass spectrometer by means of a syringe, infused at a flow rate of
3 Lmin1. The source voltage was set to 3.80 kV, to
maintain a source current of 4.60 A, the sheath gas and
auxiliary gas flow rates were set to 10 and 2 units

HPLC method
The related substances and DP analysis of the MOX samples was carried out using the EP compendial reverse phase
HPLC method A [6]. Separation of the compounds was
achieved using a Waters Nova-Pak C18, 1503.9 mm i.d.,
particle size 4 m, HPLC column. A mobile phase consisting of ammonium acetate buffer (7.7 g of ammonium acetate in 400 mL of water, pH 4.8 adjusted with acetic acid)

A. Awasthi et al.

and ACN mixed at 40:60, v/v, was eluted at a flow rate of

2.5 mLmin1 through the column maintained at 50 C, and
UV detection was performed at a wavelength of 242 nm.
Although EP monographs also recommend an additional
method (method B) for separation of the less polar impurities H to K (mostly process related byproducts), but given
the fact that DPs can be eluted using method A, no further
work was done using method B.
To acquire chromatograms, 20 L injections were made
for the analytical standard and sample solutions in diluent
(20 % H2O in ACN). The system suitability was checked by
injecting MOX system suitability EP mixture containing
trace levels of known impurities. Table 1 shows the identity
and acceptance criteria of these impurities according to the
EP.
MS and H/D exchange experiments
MOX and isolated DPs were dissolved in diluent mixture
(H2O/MeOH/ACN 15:34:51, v/v/v) at concentrations of
10 gmL1. To achieve a desired mass fragmentation, ammonium acetate was mixed in diluent at 10 mM concentration.
The exchange of the labile hydrogen atoms in MOX and
DPs by deuterium atoms was carried out by preparing
100 gmL1 solutions of the analytes in deuterated chloroform (ex nmr) and then incubation of 100 diluted solution
with deuterating agent (methanol and deuterium oxide 9:1,
v/v with 5 mM ammonium acetate, pH 7.2) for 15 min at
room temperature. The resulting solution was quickly
cooled in a dry ice/ethanol bath, and the reaction was
quenched by addition of formic acid, with further cooling
for another 5 min. The final samples were immediately
infused directly into the ESI FT-MS system.

NMR methods
1

H and 13C NMR spectra of the analytes were obtained

using a Bruker 400-MHz automated NMR spectrometer.
About 1.0 mg of each sample was accurately weighed and
diluted with 0.7 mL of CDCl3. Proton chemical shifts were
referenced to residual CHCl3 at 7.27 ppm, while carbon
shifts were referenced to CDCl3 at 77.16 ppm. All spectra
were acquired at 25 C.
Forced degradation studies
Stress studies involving hydrolytic degradation and oxidation were carried out using conditions outlined in
current guidelines [24]. Since MOX is a lipophilic compound, an accurately weighed quantity was first dissolved in 10 mL ACN to a concentration of 10 mg
mL1. Aliquots of the stock solution were spiked with
the stressor (e.g. solution of HCl, NaOH, H 2O2 or
water) and after appropriate time periods, the volumes
were adjusted with diluent (80:20 ACN/water) to obtain
a final concentration 1 mgmL1. Hydrolytic decomposition of the MOX was carried out using water and
methanolic 1 N HCl or 1 N NaOH at 70 C for 2 h.
The oxidation study was carried out in 30 % (v/v)
aqueous H2O2 at room temperature for 12 h. For all
stress conditions, parallel blank sets were treated similarly. Samples treated with acid or alkali were neutralized to pH 7.0 (using similar strength for alkali or acid
solutions used in experiments) before injecting into the
HPLC.

Results and discussion

Table 1 Moxidectin impurities and their relative retention time (RRT)
from the European Pharmacopeia (EP)
Impurity

Limit (wrt moxidectin)

a

Impurity A
Impurity Ba
Impurity Ca
Impurity Da
Impurity E+Fa
Impurity Ga
Impurity H+Ib
Impurity Jb
Impurity Kb
Any other impuritya,b
Totala+b
Disregarda,b
a

Method A

Method B

1.5 %
0.5 %
1.5 %
2.5 %
1.7 %
1.5 %
1.0 %
0.5 %
0.5 %
0.5 %
7 %
0.3

RRT
0.5
0.7
0.8
0.9
1.31.5
1.6
2.0
2.2
3.4

HPLC results
MOX standards and test samples were prepared as described in HPLC method section. The chromatogram
of the reference standard solution (system suitability
standard solution) showed an adequately separated principal peak of MOX at 12.1 min as well as EP impurities appearing at their specified relative retention time
(RRT; see Fig. 2). This method was then used for the
analysis of MOX samples under stress using the conditions outlined in Forced degradation studies section.
Thermal hydrolysis
MOX was found to be relatively unaffected by thermal
hydrolysis at 70 C for 24 h, and no known impurity listed
in EP or any DP significantly increased after treatment (see
Fig. 3).

Characterization of degradation products of moxidectin

Fig. 2 HPLC chromatogram
showing separation of the
moxidectin peak and related
substances as identified in
European Pharmacopeia

Base hydrolysis

Acid hydrolysis

As shown in the chromatogram presented in Fig. 3, reaction

with alkali resulted in a significant DP appearing before the
MOX peak (RRT 0.9), matching its retention time and UV/
vis spectrum with impurity D in the EP impurity mixture
(system suitability mix). Notably, alkaline conditions affecting the C2C5 positions in ML molecules are well documented [22, 25]. MOX (similar to ivermectin or
eprinomectin) have the 16-membered ML ring comprising
the potentially reactive hexahydrobenzofuran ring system,
which is significantly affected by the presence of an alkaline
environment.
The degradation pathway for alkaline degradation of
the MOX is identical to that proposed by Pivnichny et
al. for abamectin [25]. The reaction results in a change
in orientation of the proton attached to the C2 position
and formation of the 2-epi stereoisomer. This stereoisomer and the original substrate undergo a double bond
shift into conjugation from C3C4 to C2C3, giving
rise to the thermodynamically favoured and stable
2,3-regioisomer as the end product. Impurities D and
E presented in the EP monograph are the 2-epi- and
2,3-isomers of MOX, as shown in Table 1, and the
chromatogram from alkaline hydrolysis validates this
argument in that impurity D and E are generated. For
the MOX API, these two impurities may originate from
the manufacturing process, which involves the use of
sodium hydroxide solution to achieve the desired reaction conditions producing the final product. Nevertheless, these two impurities can also be DPs, if MOX API
and its formulations are exposed to alkaline conditions.
Since the origin, structure and degradation pathways of
the 2-epi- and 2,3-isomer are well known, no further
work was carried out for characterizing these DPs.

Acid hydrolysis typically resulting in the loss of the

terminal monosaccharide of the oleandrosyl moiety and
formation of a monosaccharide linked macrocyclic aglycone moiety is one of the most discussed chemical
reactions of MLs [26]. In the case of MOX, which is
not glycosidically linked and does not possess any oleandrosyl moieties, this route of degradation is not applicable. No investigation to date discusses the fate of
MOX under acid hydrolysis.
As shown in Fig. 3, acid-catalyzed degradation of MOX
results into two significant DPs at RRT 0.4 and RRT 1.2,
which do not resemble with any of the known impurities
present in the EP. Therefore, these two impurities were the
ideal candidates for further identification and characterization
experiments.
Oxidation
Oxidation of MOX using conditions described in
Forced degradation studies section did not cause
any significant degradation (see Fig. 3), although an
impurity at RRT 1.2, also produced by acid hydrolysis,
is present at low levels. One of the reasons MOX
shows apparent resistance to radical-induced oxidation
may be governed by the fact that commercial API
normally contains the antioxidant butylated hydroxytoluene [6], yet the susceptibility of MOX as well as its
precursor nemadectin to oxidation is well established
[27, 28]. The reported mechanism shows that general oxidation takes place at the oxahydrindene (hexahydrobenzofuran),
i.e. C2 to C12 positions of the macrocycle, via free
radicals and results in 5-oxo/8a-oxo/5,8-dioxo derivatives [29].

A. Awasthi et al.
Fig. 3 Forced degradation of
moxidectin represented by
chromatograms corresponding
to: a no treatment, b effect of
heat, c effect of acid, d effect of
alkali and e effect of oxidizing
agent on moxidectin

Isolation and purification of degradation products

The stress study sample from acid hydrolysis showing >10 %
levels of DPs (at RRT 0.4 and 1.2) was used for their isolation
and purification on a flash chromatographic system using a
glass column (70025 mm) filled with silica gel (Devisil,

63 m). A concentrated DCM aliquot was applied to the

column and eluted with diethylether/dichloromethane/methanol (9:9:1, v/v/v). The recovered solvent eluants were concentrated in vacuo using a rotary evaporator, and the resulting
chromatographically pure compounds were collected and
stored refrigerated under nitrogen prior to further analysis.

Characterization of degradation products of moxidectin

Mass spectrometry characterization of moxidectin

and its impurities
In general, MS techniques have been reported for identification of drugs and impurities using accurate mass and
fragmentation patterns [22, 3032]. LC/MS/MS procedures
described in the literature employ either triple quadrupole or
quadrupole ion trap analyzers, and the product ion mass
spectra generated by CID demonstrate significant differences regarding the presence or absence of fragment ions as
well as product ion abundances. CID fragmentation of target
analytes giving rise to major product ions has been proposed
in the literature occasionally but has not been further evaluated by systematic chemical or mass spectrometric experiments with higher resolution and accuracy. We have now
examined MOX and its DPs by comparison of accurate
mass, fragmentation pathways, multistage mass fragmentation (MSn) and H/D exchange studies using high-resolution
LTQ FT-MS. MSn and H/D exchange data are also explored
to determine the fragmentation pathways and structural
arrangements of MOX and its DPs.
MS data management programmes (HiChem MassFrontierTM and ACD/MS ManagerTM) were initially used to
predict theoretical mass fragmentation pathways. The fragmentation patterns observed were then correlated with experimental fragment data. Since MLs do not ionize well
under ESI conditions [33], 10 mM ammonium acetate was
added to the solvent mixture (MeOH/ACN/H2O, 27:48:25,
v/v/v) to assist with sample ionization.
Moxidectin
The FT mass spectrum for the molecular ion series of MOX
under 0 V in-source CID conditions is shown in Fig. 4a. The
experimental masses, theoretical masses, rings+double bonds
(RDBs), mass error [parts per million (ppm)] and consequent
atomic composition of each fragment ion are shown in Table 2.
MOX presents as a pseudomolecular ion at m/z 640.38412
and an intense sodiated adduct peak [M + Na] + at m/z
662.36684. The common fragment ions observed for MOX
preparations were m/z 622.37443, 590.34818, 572.33826,
528.29575, 510.28555, 498.28536, 496.26965, 478.25911,
452.17124, 448.28486, 434.26864, 416.25858, 220.73091,
218.15393, 199.11165 and 196.16961.
Comprehensive and elaborate MS fragmentation of
MOX was undertaken using CID at 3570 V collision
energy to generate MSn mass fragment data with concomitant HR-MS data captured when the ion intensity was sufficient (see Fig. 5). To elucidate the fragmentation scheme,
accurate masses were used to derive elemental compositions, molecular formulae and assign structures to the fragments to explain the losses. MSn studies significantly helped
to understand the origin and fragmentation pathways of the

ions found in the FT mass spectra. The molecular ion at m/z

640.38412 (MS1) undergoes fragmentation by sequential
H2O and methanol loss or by elimination of the aliphatic
side chain at the C25 position. A loss of H2O from the
pseudomolecular ion results in a dehydrated ion at m/z
622.37443 (MS2), which in turn loses methanol from the
methoxime group to give rise to an ion at m/z 590.34818
(MS3). Further H2O loss gives an aromatic ion at m/z
572.33826 (MS4), followed by formation of m/z
554.32668 (MS5). On other hand, loss of the aliphatic side
chain at C25 (C 7 H 12 O) results in a key ion at m/z
528.29575 (MS2), which produces three different ions by
elimination.
1. Loss of water from this ion results in an ion at m/z
510.28555 (MS3), which then loses methanol and produces an ion at m/z 478.25911 (MS4).
2. Alternatively, loss of HCOH from m/z 528.29575 gives
rise to an ion at 498.28536 (MS3), where an opening of
the macrocycle at the C2C19 position produces an ion
at m/z 454.29196 (MS4).
3. On the other hand, a loss of methanol from the same key
ion produces an ion at m/z 496.26965 (MS2), which in
turn loses CO2, and a subsequent loss of water from this
ion results in formation of an ion at m/z 434.26864
(MS5) followed by 416.25858 (MS6). The latter ion
undergoes allylic cleavage and produces ions at m/z
199.1165 and 218.15391 (MS7). These two ions confirm the structure and groups present at key functional
positions like C2C5 and C19C24 positions. The
breakdown and tracking of ions up to MS7 levels demonstrate the power of this method in verifying the structural arrangement of MOX-type molecules.
Impurity 1 (RRT 1.2)
The FT mass spectrum for the molecular ion series of
impurity 1 under 35 V in-source CID conditions to induce
a degree of incipient fragmentation is shown in Fig. 4b. The
experimental and theoretical masses, RDBs, mass error
(ppm) and atomic composition of the molecular ion series
are listed in Table 3. The FT mass spectrum exhibits a
pseudomolecular ion at m/z 656.37952 and an intense sodiated adduct peak [M+Na]+ at m/z 678.36224, which is
exactly 15.99540 Da more than the corresponding MOX
mass. Comparison of the accurate masses indicates an additional O atom and the possibility of the presence of an
epoxy derivative of moxidectin. Except for a few smaller
key ions, the common fragments observed for the molecular
ion under 35 V in-source CID also showed 16 Da additions
to fragments previously seen with MOX fragmentation. The
main daughter ions observed for impurity 1 were m/z
638.36902, 606.34318, 544.29071, 544.29071, 526.28053,

5.0

662.36684

640.38412

622.37443

498.28536

Atul_Moxi_1 #1-1632 RT: 1.50-1.73 AV: 16 NL: 1.55E7

F: FTMS + c ESI Full ms [100.00-1000.00]

a. Moxidectin

638.37030
590.34818

620.35579
624.38109

0.5

537.87948 530.30288

1.0

479.26223

163.63426

1.5

393.02062

196.16961
218.15391
220.73091

2.0

417.26384

2.5

448.28486
449.28822
459.23808

416.25858

3.0

664.37362

3.5

643.39441

478.25911

4.0

499.28873

4.5

Relative Abundance

Fig. 4 FTMS of: a moxidectin

(0 V), b impurity 1 (35 V), c
impurity 2 (60 V) under variable
in-source CID conditions (see
text) to induce incipient fragmentation of the respective molecular ions to provide an
overview and sufficient daughter
ions for reliable accurate mass
determination ([M+H]+ peak for
impurity 2 is shown in inset)

528.29575

A. Awasthi et al.

0.0
150

200

250

300

350

400

450

500

550

600

650

679.36530

678.36224

656.37952

b. Impurity 1
639.37275

22

658.38665
663.45452

640.37618

636.35375

606.34318
607.34626

482.29036

545.29417
538.77478
546.29710

414.24296

359.18848

288.28967

269.39322

215.10672
218.15397
220.70789

432.25354
433.25686

10

483.29385

12

453.28318

14

450.26415
452.27972

16

495.25740
496.26942
515.28359

Relative Abundance

18

494.25400
512.26457
514.28036

20

680.36852
688.37159

24

544.29071

Atul_Moxi_34oxo_5 #9-261 RT: 0.19-0.49 AV: 21 NL: 7.49E6

F: FTMS + c ESI sid=35.00 Full ms [185.00-1000.00]

638.36902

m/z

0
200

250

300

350

400

450

500

550

600

650

m/z

24

633.33694

593.34585

575.33512

23-keto-Moxi_1 #1-3387 RT: 31.04-31.28 AV: 17 NL: 4.00E6

F: FTMS + p ESI sid=60.00 Full ms [130.00-1000.00]

c. Impurity 2

22

644.35565

615.32896

622.37323

590.34723

557.32584

513.33603

405.20622

389.21127

363.19556

323.16417

345.18499

277.15873

297.14858
305.15364

251.14311

219.13803

10

451.21159

437.19341

12

572.33675

14

528.29553

481.25816

16

637.30489

18

199.11182

Relative Abundance

20

0
150

200

250

300

350

400

450

500

550

600

650

m/z

514.28036, 512.26457, 494.25400, 482.29036, 468.27461,

464.27982, 452.27972, 450.26415, 434.26898, 432.25354,

414.24296, 359.18848, 218.15397, 215.10672 and

196.16973.

Characterization of degradation products of moxidectin

Table 2 FTMS analysis of
moxidectin: theoretical and experimental mass, detection accuracy, rings+double bonds
(RDBs) equivalent, chemical
composition and exchangeable
hydrogen data

m/z

Theoretical
mass

Delta
(ppm)

RDB equivalent

Composition

H/D exchange
Increase

662.36684
640.38412
622.37443
590.34818
572.33747
510.28555
498.28536
496.26965
478.25911
452.17124

662.36634
640.38439
622.37383
590.34761
572.33705
510.28501
498.28501
496.26936
478.25880
452.17038

0.50
0.27
0.60
0.57
0.42
0.54
0.35
0.29
0.31
0.86

11.5
11.5
12.5
13.5
14.5
11.5
10.5
11.5
12.5
13.5

C37 H53
C37 H54
C37 H52
C36 H48
C36 H46
C30 H40
C29 H40
C29 H38
C29 H36
C25 H26

O8
O8
O7
O6
O5
O6
O6
O6
O5
O7

448.28486
434.26864
416.25858
218.15391
199.11165
196.16961

448.28462
434.26897
416.25841
218.15394
199.11174
196.16959

0.24
0.33
0.17
0.03
0.09
0.02

11.5
11.5
12.5
5.5
7.5
2.5

C29 H38
C28 H36
C28 H34
C14 H20
C14 H15
C12 H22

O3 N
O3 N
O2 N
ON
O
ON

Comprehensive and elaborate MS fragmentation pattern for impurity 1 was undertaken using CID at 35
70 V collision energy to generate MSn mass fragments
in the ion trap with concomitant HR-MS data obtained
when ion intensity was sufficient (see Fig. 6). To elucidate the fragmentation scheme, accurate masses and
structure of the fragments were used to derive molecular
formulae and assign elemental composition to the losses. MSn studies clearly help to understand the origin
and pathways of ions found in FT mass spectra
(Fig. 4b).
As previously seen in MOX fragmentation pattern,
the impurity 1 pseudomolecular ion at m/z 656.37952
(MS1) also undergoes unique fragmentation pattern involving loss of H2O and methanol or by cleavage of the
aliphatic side chain at C25 position. A loss of H2O
from the pseudomolecular ion results in a dehydrated
ion at m/z 638.36902 (MS2), which in turn loses methanol from the methoxime group and gives rise to an ion
at m/z 606.34318 (MS3) that undergoes McLafferty
rearrangements, leading to an opening of the macrocycle
at C2C19 position and producing an ion at m/z
562.35363 (MS4). Loss of H2O from this ion gives an
ion at m/z 544.29071(MS5), which gives rise to strategically important ions at m/z 215.10672 and 218.15397
(MS6/7), laying down strong support for the presence of
the epoxy group at C3C4 position.
On the other hand, loss of the aliphatic side chain at
C25 (C7H12O) results in a key ion at m/z 544.29071
(MS1), which further produces three different ions by
cleavage of the methoxime group with a pattern of ions

N Na
N
N
N
N
N
N
N
N
N

Accurate mass

1
1
1
1
1
1
2
2
1
3

663.37086
641.38921
623.37934
590.35309
573.34229
511.29171
500.29692
498.27954
479.26593
455.18492

1
2
2
1
1
0

449.28761
436.28363
419.27733
219.16027
200.11515

already seen (16 Da) for MOX. Again, abundance of

terminal ions, i.e. m/z 215.10672 and 218.15397 (MS7),
strengthens the argument for 3,4-epoxy-moxidectin.
Impurity 2 (RRT 0.4)
The FT mass spectrum and for molecular ions for
impurity 2 is shown in Fig. 4c under 60 V in-source
CID conditions. The experimental masses, theoretical
masses, RDBs, mass error (ppm) and possible molecular
formulae of molecular ion masses are listed are in
Table 4. This compound exhibits a minute pseudomolecular ion at m/z 611.35871 and an intense sodiated
adduct peak [M+Na]+ at m/z 633.33694 as well as a
dehydrated ion at m/z 593.34585. Experimental data
indicate the absence of a key heteroatom, i.e. nitrogen,
as well as removal of one carbon along with several
hydrogens. The combination of these features and the NMR
data for this unknown compound indicate the absence of the
methoxime group (>0NOCH3) and the presence of a keto
group (>0O) at the C23 position of MOX.
The main daughter ions observed for impurity 2
under 60 V in-source CID conditions were m/z
593 .34585 , 5 75.335 12, 557. 32584, 5 31.346 91,
513 .33603 , 4 99.267 99, 495. 27437, 4 81.258 16,
46 9.22 219 , 46 3.24 803 , 451 .2115 9, 4 45.2 375 3,
43 3.20 107 , 41 9.25 824 , 389 .2112 7, 3 63.1 955 6,
345 .18499 , 3 23.164 17, 309. 27888, 3 05.153 64,
2 77 .1 58 73 , 25 1. 1 43 11, 2 25 .0 91 0 6, 19 9. 111 82 ,
193.12237, 171.11690, 159.11680 and 151.11208. Furthermore, some unique ions were also identified during

A. Awasthi et al.
O
H3C
H 3C

N
CH3

H
O

CH 3

O
H 3C

CH3 H C
3

H
O

HO

Moxidectin
(639.37712)
O

CH 3

H
OH

Protonation

H3 C

H3 C

HO

H3C

CH3 H3C

HO

MS1

m/z640.38412
(640.38439)
H

CH3

CH3 H3C

O
O

CH3

O
CH3

m/z640.38412
(640.38439)

CH 3

CH3

H3C

NH2

H 3C

O
H3C

OH 2

RetroDiels-Alder

CH3
OH
-C7 H12O

-H2O
O
H3 C
O
H3C
H3C

H 3C

m/z622.37443)
(622.37383)

CH3
O
CH 3

O
H 3C

NH2
CH3

HO

H3C

CH 3 H3C

m/z528.29575
(528.29613)

HO

CH3

H
O

-H2 O

H 3C

O
CH3

O
O

HO

NH2

H 3C

H3C

m/z510.28555
(510.28501)

HO

H
m/z498.28536
(498.28501)

HO

H 3C

CH3 H3C

H3C

H3 C

m/z572.33826
(572.33760)

HO

m/z452.17124
(452.28008)

HO

CH3

OH

-H2 O

OH

NH
H

McLafferty
Rearrangement
NH

H 3C

CH3
O

H3C
CH3

MS5

-CO2
HO

O
CH3

m/z434.26864
(434.26897)

CH3 H 3C

H3C

CH3
O

MS4

H
CH3

H 3C
-H 2O

NH

-CO2

H3C

m/z454.29196
(454.29573)

CH3

CH3

CH3
O

O
O

CH3

NH 3

MS3

HO

m/z478.25911
(478.25880)

OH

H 3C

H 3C

McLafferty
Rearrangement

-CO2

CH3

O
HO

-CO2

CH 3

H3C

CH3

m/z496.26965
(498.26936)

OH

McLafferty
Rearrangement

NH

NH
H

H3C

CH 3

CH 3

-CH3 OH

H3C

O
O

-H2O

CH3

H3C

CH 3

H3 C

CH 3

CH 3

CH3 H3C

NH

NH3

O
H3C

CH3

H3C

-CH3OH

-CHOH

NH

m/z590.34818
(590.34761)

OH

CH3

-CH3OH

H3 C

MS2

CH3

O
-H2 O

m/z554.32668
(554.32703)

NH

H3 C
O

CH3

CH3
O

MS6

H3C
m/z416.25858
(416.25841)
O

CH3

AllylicCleavage

NH
H3C

m/z199.11165
(199.11174)

H3 C

CH3
O
MS7

CH 3

m/z218.15391
(218.15394)

Fig. 5 MSn fragmentation pathway of moxidectin showing ions with accurate masses and structural arrangements (bracketed values represent
calculated m/z values)

Characterization of degradation products of moxidectin

Table 3 FTMS analysis of Impurity 1: theoretical and experimental mass, detection accuracy,
rings+double bonds (RDBs)
equivalent, chemical composition and exchangeable hydrogen
data.

m/z

Theoretical mass Delta (ppm) RDB equivalent Composition

H/D exchange
Increase Accurate mass

678.36224
656.37952
638.36902
606.34318
562.35363
544.34250
544.29071
526.28053
514.28036
512.26457

678.36180
656.37986
638.36929
606.34308
562.35325
544.34268
544.29104
526.27993
514.28048
512.26483

0.44
0.34
0.27
0.10
0.38
0.18
0.33
0.60
0.12
0.26

11.5
11.5
12.5
13.5
12.5
13.5
10.5
11.5
10.5
11.5

C37 H53 O9 N Na
C37 H54 O9 N
C37 H52 O8 N
C36 H48 O7 N
C35 H48 O5 N
C35 H46 O4 N
C30 H42 O8 N
C30 H40 O7 N
C29 H40 O7 N
C29 H38 O7 N

1
1
1
1
2
1
1
1
2
2

679.36681
657.38482
639.37412
607.34773
564.35995
546.34698
545.29586
527.28648
516.29298
514.27946

494.25400
482.29036
468.27461
464.27982
452.27972
450.26415
434.26898
432.25354
414.24296
359.18848
218.15397
215.10672
196.16973

494.25426
482.29065
468.27500
464.28008
452.28008
450.26443
434.26952
432.25387
414.24330
359.18853
218.15449
215.10720
196.17014

0.26
0.29
0.39
0.26
0.36
0.28
0.54
0.33
0.34
0.05
0.52
0.48
0.41

12.5
10.5
10.5
11.5
10.5
11.5
11.5
12.5
13.5
13.0
5.5
7.5
2.5

C29
C29
C28
C29
C28
C28
C28
C28
C28
C24
C14
C14
C12

1
2
2
2
2
2
1
1
2
1
1
0
1

495.26103
484.29997
470.28808
466.2898
454.28936
452.28014
435.27614
433.26044
416.25887
360.19356
219.17447

197.17598

the in-source CID experiments on the protonated and

sodiated molecular ion peaks.
Comprehensive and elaborate MS fragmentation analysis of
impurity 2 was undertaken using CID at 3570 V collision
energy to generate MSn mass fragment data with concomitant
HR-MS when the ion intensity was sufficient (see Fig. 7). To
elucidate the fragmentation scheme, accurate masses and structure of the fragments were used to derive molecular formulae
and assign elemental compositions to the losses. Impurity 2
produces the molecular ion peak with a mass 29.02728 Da
lower than MOX. Apart from a typical water/alcohol loss
series, depending on the cleavage position whether at C23 or
C24, it produces two series of fragmentations, providing the
key ions to support the proposed structure for impurity 2.
A loss of H2O molecule from parent molecular ion results
into a dehydrated ion at m/z 593.34711 (MS2), which facilitates the opening of macrocycle at C1C19 position by loss of
an H2O molecule and gives rise to an ion at m/z 575.33646
(MS3). This ion further undergoes losses of water and CO to
give rise to ions at 557.32619 (MS4), m/z 531.34691 (MS5)
followed by m/z 513.33634 (MS6).
On the other hand, loss of the aliphatic side chain at C25
(C7H12O) results in a key ion at m/z 499.26799 (MS2),
which upon losing H2O molecules produces ions at m/z

H36
H40
H38
H38
H38
H36
H36
H34
H32
H25
H20
H15
H22

O6 N
O5 N
O5 N
O4 N
O4 N
O4 N
O3 N
O3 N
O2 N
O2 N
ON
O2
ON

481.25849 (MS3), 463.24803 (MS4) and 445.23753 (MS5).

From here, allylic cleavage leads to the formation of an ion at
m/z 419.25824 (MS6). This ion on fragmentation produces
key ions at m/z 219.13783, 199.11179 and 151.11208 (MS7),
providing further support to the proposed identity of the parent
compound as 23-keto-nemadectin. Similarly, a cleavage taking place at C23 produces ions similar to the C25 cleavage
series, providing further evidence for this structure.
HX-MS studies
Due to the back exchange problem associated with the commonly used online exchange method, hydrogen/deuterium
exchange was carried out by a chilling and quenching process
as described in Methods section, and spectra were recorded
by FT-MS. The ions (as shown in Table 2, 3 and 4) possessing
labile hydrogen underwent exchange process. The pseudomolecular ion peaks for MOX, as well as impurities 1 and 2,
showed an increase of one m/z unit corresponding to one
exchangeable hydrogen present at C5OH. Sequential loss
of water molecules as well as opening of the macrocycle
making C7OH available for exchange was observed for all
three compounds under investigation, further supporting the
proposed structures of MOX as well unknown compounds.

A. Awasthi et al.
O
H3C
H3C

N
CH3

H
O

CH3

O
H3C
O

HO

3,4-oxo-moxidectin
(655.37203)

CH3 H C
3

O
O

CH3

H
OH

Protonation
O
H3C
H3C

O
H3 C
H3C

HO

H3C

CH3
CH3 H3C

m/z656.37952
(656.37986)

HO

H
O
CH3

CH3

OH

-C7H12O

OH2

RetroDiels-Alder
O

-H2O

H3C
H3 C

O
H3 C
H3C

NH
CH3

H3C

O
CH3

O
H3C

m/z638.36902)
(638.36929)

MS1

CH3 H C
3

O
O

CH3

O
O

m/z656.37952
(656.37986)

CH3

H3C

NH
CH3

HO

m/z544.29071
(544.29104)

CH3H3C

NH2
CH3
O

HO

MS2

O
CH3

O
H

OH

O
O

CH3

H3C

-CHOH

NH
H

H3 C

m/z606.34318
(606.34308)

NH
O
H3 C

CH3
O

H3C

HO

CH3

H3C

CH3 H C

O
H

m/z526.28053
(526.27993)

O
O

CH3
O

HO

H3 C

H3C

O
CH3

CH3

HO

H3 C

H3 C

H3 C
H

H
m/z470.28808
(470.29573)

O
CH3

MS4

O
H

OH

HO
O

m/z468.27461
(468.27500)

NH

H3C

CH3

O
CH3

CH3
O

CH3

MS5

H 3C

CH3H3C

H3C
HO
m/z450.26415
(450.26443)

HO

O
O

m/z452.27972
(452.28008)
m/z544.34250
(544.34268)

NH

H3C

O
O

OH

-H2O

CH3

CH3

O
CH3

NH3

H3 C

-H2O

CH3

CH3

HO
m/z494.25400
(494.25426)

O
CH3

-H2O

H3C

CH3

McLafferty
Rearrangement
NH

H3 C

NH

MS3

OH

-CO2

CH3

H3C

HO
m/z562.35363
(562.35325)

CH3
CH3H3C

H3 C

O
H

-CH3OH

O
O

O
HO

m/z512.26457
(512.26483)

NH3
NH

CH3
O

CH3
OH
McLafferty
Rearrangement

-CO2

-CO2

H3C
H3C

m/z514.28036
(514.28048)
H

H3C

NH2

HO

O
CH3

CH3
O

H3C

-CH3OH

-H2O
NH3

-CH3OH

O
CH3

CH3
NH

-H2O
H3C

CH3

MS6

H3 C
m/z432.25354
(432.25387)
O
CH3

AllylicCleavage
RetroDiels-Alder

NH
H3C

m/z215.10672
(215.10720)

H3C

O
CH3

CH3
O

MS7

m/z218.15397
(218.15449)

Fig. 6 MSn fragmentation pathway of impurity 1 showing ions with accurate masses and structural arrangements (bracketed values represent
calculated m/z values)

Characterization of degradation products of moxidectin

Table 4 FTMS analysis of impurity 2: theoretical and experimental mass, detection accuracy,
rings+double bonds (RDBs)
equivalent, chemical composition and exchangeable hydrogen
data.

m/z

Theoretical mass Delta (ppm) RDB equivalent Composition

H/D exchange
Increase Accurate mass

633.33694
611.35871
593.34585
575.33512
557.32584
531.34691
513.33603
499.26799
495.27437
481.25816

633.33679
611.35839
593.34728
575.33672
557.32615
531.34689
513.33632
499.26958
495.27412
481.25847

0.15
0.32
1.43
1.60
0.31
0.02
0.29
1.59
0.25
0.31

11.5
11.5
12.5
13.5
14.5
12.5
13.5
10.5
11.5
11.5

C36
C36
C36
C36
C36
C35
C35
C29
C30
C29

H50 O8 Na 1
H51 O8
1
H49 O7
1
H47 O6
2
H45 O5
0
H47 O4
1
H45 O3
1
H39 O7
1
H39 O6

H37 O6
2

634.34448
612.36085
594.35237
577.34675

532.34994
514.33913
500.27251

483.26836

469.22219
463.24803
451.21159
445.23753
433.20107
419.25824
415.21277
389.21127
305.15364
277.15873
251.14311
225.09106
219.13803
199.11182
193.12237
171.11690
159.11680
151.11208

469.22208
463.24790
451.21152
445.23734
433.20095
419.25807
415.21152
389.21112
305.15361
277.15869
251.14304
225.09101
219.13796
199.11174
193.12231
171.11683
159.11683
151.11174

0.11
0.13
0.07
0.19
0.12
0.17
1.25
0.15
0.03
0.04
0.07
0.05
0.07
0.08
0.06
0.07
0.03
0.34

11.5
12.5
12.5
13.5
13.5
11.5
9.5
12.5
11.5
10.5
9.5
9.5
5.5
7.5
4.5
6.5
5.5
3.5

C27
C29
C27
C29
C27
C28
C24
C26
C21
C20
C18
C15
C14
C14
C12
C13
C12
C10

H33 O7
H35 O5
H31 O6
H33 O4
H29 O5
H35 O3
H31 O6
H29 O3
H21 O2
H21 O
H19 O
H13 O2
H19 O2
H15 O
H17 O2
H15
H15
H15 O

470.22908
464.25039
453.22472

434.20820
420.26120

220.14218

NMR studies
As part of this study, moxidectin and unknowns (impurities 1
and 2) were fully characterized by NMR spectroscopy.

1
1
2
0
1
1

1
0

detailed interpretation results leading to assignment of

the structure and refer to Electronic supplementary material
(ESM) Fig. S1 for detailed NMR spectrums of MOX.
Impurity 1 (RRT 1.2)

Moxidectin
The 1H and 13C NMR chemical shifts of moxidectin
were consistent with those reported by Rajan and Stockton for nemadectin (LL-F28249) [34]. The only significant differences in the 1H NMR data reported for
nemadectin and those obtained for moxidectin were the
presence of a methoxy peak at 3.83 in the latter and
the large downfield shift of one H22 ( 3.28), consistent
with an adjacent oxime (in place of the carbinol). The
carbon resonances were also very similar except at C23,
which is a secondary alcohol in nemadectin ( 69.13)
and an oxime ( 156.1) in moxidectin. See Table 5 for

Impurity 1 differs from its precursor, moxidectin, in the

resonances around the hexahydrobenzofuran moiety. Most
notably, olefinic resonances for C3 (H 5.41, C 118.3) and
C4 ( 138.0) are replaced by oxygenated aliphatic signals
(H 3.38, C 59.8 and 59.5). Slight differences at positions 2,
4a, 5a, 7, 8 and 8a indicate that different chemical environments are present and an altered conformation is adopted by
the bicyclic system. For the remainder of the molecule, differences in the spectral signals are minimal. See Table 6 for
detailed interpretation results leading to assignment of the
structure and refer to ESM Fig. S2 for detailed NMR
spectrums of impurity 1.

A. Awasthi et al.
O
H3 C

CH3

H
O

CH 3

O
H3C

HO

CH3 H C
3

23-keto-nemadectin
(610.35057)
O

CH3

H
OH

P rotonation

O
CH3
H3 C

m/z 611.35871
(611.35839)

HO

CH3

CH 3

H3C

m/z 611.35871
(611.35839)
CH 3

H3C

HO

H3 C

CH3

HO

m/z .499.26799
(499.26958)

m/z 469.22219
(469.22208)

CH3

HO

CH3

OH
Allylic Cleavage

O
H3C

H 3C

CH 3

MS3
H3 C

CH3

H3C

O
HO

CH3H 3C

CH3

O
O

OH

HO

m/z 451.21159
(451.21152)

HO

O
m/z 481.25849
(481.25847)

CH3

CH 3

CH3
OH

-H2O
O

-H 2O

-H 2O

H 3C

O
CH3

-H2O

CH3

OH

A llylic Cleavage

m/z 575.33646
(575.33672)

H3C

H
OH

CH3 H C

m/z 593.34711
(593.34728)

H3C

-C4H17 O

MS2

O
O

OH

CH3

CH3

H 3C

CH3

Allylic Cleavage
H 3C

-H2 O

MS1

O
H3C

H3 C

m/z 611.35871
(611.35839)

CH H
3 3C

O
H

-H2 O

H3C

O
HO

OH
-C7H 12O

Allylic Cleavage

O H2

CH 3

O
H

CH 3

CH3
O

CHH
3 3C

H
OH
H

O
HO

CH3H3 C

CH3

O
H

CH3
O

O
O

H 3C

CH 3

H 3C

CH3

MS4

H3 C

CH3

CH3
O

OH

CHH
3 3C

H 3C

H3 C
O

m/z 557.32619
(557.32615)
O

m/z 463.24803
(463.24790)

CH3

m/z 433.20107
(433.20095)

HO

H 3C

-H2O

OH
H

CH3

-CO

CH3

CH 3

-COOH

CH3

CH3

CH 3

CH3

H3 C

MS5

CHH33C

H3 C

H3 C
m/z 531.34691
(531.34689)

m/z 445.23753
(445.23734)

CH 3

m/z 389.21115
(389.21112)
O

CH 3

CH 3
Allylic Cleavage

-H 2O
-CO
CH 3

CH3

CH3

CH
H33C

H3C
m/z 513.33634
(513.33632)

OH

OH
CH 3

H2 C

CH3

HO

H3C

H3C
O

m/z 199.11179
(199.11174)

m/z 419.25824
(419.25807)

CH 3

CH 3
Allylic Cleavage

H3 C
O

CH 3

MS 6

m/z 193.12237
193.12231

OH
CH3

H2C

H 3C

H 2C

H
O

H2C
O
m/z 151.11208
(151.11174)

O
H

MS7
CH3

CH3

m/z 199.11179
(199.11174)

m/z 219.13783
(219.13796)

Fig. 7 MSn fragmentation pathway of impurity 2 showing ions with accurate masses and structural arrangements (bracketed values represent
calculated m/z values)

Characterization of degradation products of moxidectin

Table 5

13

C and lH NMR chemical shift assignments and correlation data for moxidectin
O

30

H3C

24a

14a

CH3
14
13
12a

16

10

HO
8

1
2

O
H

Position
1

C (ppm)

13

H (ppm)

CH3

20

28a

27

25
26

CH3
26a

28

CH3

CH3

29

H
3

8a
5a

21

O
19

18

11

H3C

17

15
12

23 24

22

4a

CH3

OH

Integration

Multiplicity

Coupling J (Hz)

H1H COSY

HMBC 1H to

13

2.3

H3, H5

C1, C3

H2, H4a

C2, C4a, C5, C8a

C3, C4, C5

173.8

45.9

3.27

118.3

5.41

sharp m

138.0

4a

20.1

1.87

1.6

H3

67.8

4.28

br. t

7.1

H2, H5a, OH-5

C3, C4

5a

79.4

3.95

6.2

H5

C2, C4, C8, C8a

7a
7b

68.6

4.71
4.65

1
1

dd
dd

14.3, 2.0
14.3, 2.0

H9

C5a, C8, C9

139.6

8a

80.4

120.5

5.78

dt

11.2, 2.3

H7, H10

C7, C8a

10

123.5

5.73

dd

13.6, 11.3

H9, H11

C12

11

143.0

5.33

obs. m

H10, H12

C9, C12

12

36.09

2.41

H11, H12a, H13b

12a

22.4

0.99

H12

C11, C12, C13

13a
13b

48.6

2.18
1.86

1
1

obs. m
obs. m

H12, H13b, H14a

H12, H13a

C11, C12, C14

C12, C12a, C14, C14a, C15

14

137.1
H13a, H15

C13, C14, C15

6.6

14a

15.7

1.51

15

121.0

4.92

dd

16

34.5

2.21

obs. m

17

68.3

3.47

18a
18b

36.05

1.78
0.86

1
1

m
q

19

68.5

5.33

obs. m

20a
20b

40.8

2.15
1.41

1
1

21

98.8

22a
22b

35.7

3.28
1.91

1
1

23

156.1

24

37.6

2.30

H24a, H25

C23, C24a, C25

24a

11.1

0.91

6.7

H24

C23, C24, C25

25

81.9

3.62

10.3

H24

C23, C24a, C26, C26a, C27

26

130.5
C25, C26, C27

10.1, 5.1

H16

C13, C14a

H15, H17

C14, C15, C17, C18

H16, H18b

11.8

H18b, H19
H18a, H17, H19

C19, C20
C17 and/or C19

H18a, H18b, H20a, H20b

C1

obs. m
t

11.8

H19, H20b
H19, H20a

C19, C21
C19, C21

d
d

14.6
14.9

C21, C23, C24

C21, C21, C23

26a

11.0

1.65

1.2

H27

27

138.2

5.17

dd

9.0, 1.2

H26a, H28

C25, C26, C28, C29, C30

28

27.0

2.59

H27, H29, H30

C27, C29

29

22.9

0.96

6.7

H28

C27, C28, C30

30

23.0

1.04

6.6

H28

OCH3

61.5

8.6

H5

3.83

OH-5

2.35

OH-8a

3.87

C5
C2, C5a, C8a

A. Awasthi et al.
Table 6

13

C and lH NMR chemical shift assignments and correlation data for impurity 1: 3,4-epoxy-moxidectin
O

30

H3C

24a

14a

CH3
13
12a

18

11

HO

H (ppm)

CH3

CH3

26a

29

4
5

CH3

28

C (ppm)

20

28a

27
26

8a
5a

13

CH3

25

9
8

Position

21

19

10

17

15
12

H3C

23 24

22

16

14

CH3
4a

OH

Integration

Multiplicity

170.0

45.7

3.19

59.8

3.38

59.5

Coupling J (Hz)

H1H COSY

HMBC 1H to 13C

C1, C8a
1.0

C2, C3, C8a

4a

19.5

1.51

obs. s

68.3

4.27

ddd

6.7, 5.2, 1.0

OH-5, H-6

C3 and/or C4

5a

81.9

3.90

6.7

H-5

C2, C4, C8a

67.7

C3 and/or C4, C5

4.53

dd

13.4, 2.3

H-9

C8, C9 or C10

4.65

dd

13.4, 2.0

H-9

C8

137.0

8a

77.4

123.8

5.89

app. dt

11.4, 2.2

H-7, H-10

10

123.8

5.77

dd

14.6, 11.4

H-9, H-11

11

143.6

5.39

dd

14.7, 10.0

H-10, H-12

12

36.3

2.43

12a

22.4

0.99

6.7

H-12

C11, C12, C13

13a
13b

48.5

1.88
2.22

1
1

obs. m
obs. m

H-12, H-13b
H-13a

C12, C14a, C15

C14, C15

14

137.2

14a

15.7

1.51

obs. s

15

121.3

4.95

br. dd

16

34.3

2.22

17

68.5

3.47

18a

35.6

0.86
1.89

18b

H-11, H-12a, H-13a

H-15

C13, C14, C15

H-14a, H-16

C14a

obs. m

H-15, H-17

C15, C17

obs. m

H-16, H-18a

obs. m

H-18b, H-17, H-19

obs. m

H-18a, H-19

10.5, 4.9

C17 and/or C19

19

68.8

5.24

20a
20b

41.1

1.31
2.31

1
1

t
obs. m

11.5

H-18, H-20
H-19, H-20b
H-19, H-20a

21

98.8

22a
22b

35.6

1.90
3.27

1
1

d
d

14.9
14.6

H-22b
H-22a

23

156.3

24

37.5

2.32

obs. m

H-24a, H-25

C23

24a

11.0

0.91

6.6

H-24

C23, C24, C25

25

81.5

3.62

10.3

H-24

C24a and/or C26a, C27

26

130.6
1.2

C19

26a

11.1

1.65

H-27

C26, C27

27

138.2

5.17

H-26a, H-28

C25, C26a

28

27.0

2.60

H-27, H-29, H-30

29

23.0

0.96

6.7

H-28

30

23.0

1.05

6.7

H-28

OCH3

61.5

3.83

OH-5

2.88

OH-8a

3.93

3.51

MeOH

51.0

C27, C28, C30

C27, C28, C29
C23

5.2

H-5

C3
C8

0.96

Characterization of degradation products of moxidectin

Table 7

Position
1

13

C and lH NMR chemical shift assignments and correlation data for impurity 2: 23-keto-nemadectin

13

C (ppm)

H (ppm)

Integration

Multiplicity

Coupling J (Hz)

H1H COSY

HMBC 1H to

13

173.7

45.9

3.26

H3, H4a

118.2

5.41

sharp m

H2, H4a

138.1

4a

20.1

1.88

br. s

H3

67.8

4.29

br. m

H4a, H5a

5a

79.4

3.96

68.6

4.68

complex m

139.5

6.2

C3, C4

H5
H9

8a

80.4

120.6

5.78

dt

11.3, 2.1

H7, H10

10

123.5

5.74

dd

13.7, 11.3

H9, H11

11

143.1

5.35

obs. m

H10, H12

12

36.1

2.43

H11, H12a, H13

12a

22.4

1.00

13a
13b

48.6

2.22
1.88

1
1

obs. m
obs. m

14

137.4

6.6

H12
H12, H13b
H12, H13a

14a

15.7

1.51

15

120.5

4.94

dd

H13b, H15

16

34.5

2.19

obs. m

H15, H17

17

68.6

3.50

obs. m

H16, H18b

18a
18b

35.9

1.84
0.88

1
1

obs. m
obs. m

H18b, H19
H17, H18a, H19

19

68.1

5.38

obs. m

20a
20b

40.6

2.27
1.43

1
1

obs. m
t

11.8

21

100.9

22a
22b

51.7

2.52
2.48

1
1

d
d

13.6
13.9

23

206.7

24

46.8

2.47

obs. m

24a

9.4

0.86

6.6

H24

25

82.1

3.72

10.3

H24

26

129.9

10.5, 5.1

C14

H14a, H16

C17 and/or C19

C17 and/or C19

H18a, H18b, H20a, H20b

H19, H20b
H19, H20a

C19
C19

H24a, H25

26a

10.9

1.70

1.2

H27

27

138.5

5.20

dq

9.1, 1.2

H24a, H28

C27

28

27.1

2.59

29

22.9

0.96

6.7

H28

C27

30

22.9

1.06

6.7

H28

C27

OH-5

2.35

br. d

7.3

H5

OH-8a

3.79

br. m

H27, H29, H30

A. Awasthi et al.

Impurity 2 (RRT 0.4)

Impurity 2 retains the chemical shift positions and patterns of the parent compound throughout the macrocyclic
and hexahydrobenzofuran regions, differing significantly
only at C23 and the flanking positions. Thus, for MOX,
the chemical shift of C23 is 156.1, consistent with an
oxime, whereas for impurity 2, this carbon is at 206.7,
typical of a ketone. The two alpha carbons, C22 and
C24, are shifted downfield in impurity 2. Interestingly,
the protons at C22 are less differentiated in impurity 2 (
2.52 and 2.48) than in moxidectin ( 3.28 and 1.91). See
Table 7 for detailed interpretation results leading to assignment of the structure and refer to ESM Fig. S3 for detailed
NMR spectrums of impurity 2.
Mechanism of the MOX degradation pathways to impurity 1
and impurity 2
Impurity 1 (RRT 1.2)
The formation of epoxides under the influence of mild acidic
conditions has been reported as a possible derivatization of
MLs [35]. This is a typical acid (possessing mild oxidizing
properties, e.g. m-chloroperbenzoic acid) catalyzed oxidation
of the oxahydrindene unit of MOX. Literature reviews indicate the formation of a single or a mixture of several epoxides
at the C3C4, C10C11 and C14C15 olefinic double bonds

[35]. An accurate mass of the impurity 1 molecular ion peak

by FT-MS shows a value at m/z 656.37952 (16 Da addition to
MOX) indicating the presence of a monoepoxide, and subsequent MSn fragmentation pathways, H/D exchange and NMR
data confirm the presence of this epoxide at the C3C4 position of MOX as shown in Fig. 8.
Impurity 2 (RRT 0.4)
The relationship between impurity 2, i.e. the 23-keto derivative, and the parent compound MOX, is an integral part of
the semi-synthesis of MOX, which can be understood by
exploring the steps involved in reaction sequence (Fig. 9)
[36]. As a matter of fact, the 23-keto derivative is the
penultimate compound in the reaction process, and
undergoes oxime formation at C23 under alkaline conditions
to produce MOX. During the acid hydrolysis under stress
study, reversal of the same reaction evidently occurs. Such
reactivity is well precedent for oximes under aqueous
acidic conditions. Combining the MS and NMR experimental data confirms impurity 2 as 23-keto-nemadectin
(see Fig. 8).

Conclusions
From the above studies, it is concluded that the EP method
for MOX API deals principally with the process-related

Fig. 8 Schematic diagram

showing acid-catalyzed degradation of moxidectin into 3,4epoxy-moxidectin and 23-ketonemadectin

Moxidectin
Acid, H2O

3,4-epoxy-moxidectin

23-keto-nemadectin

Characterization of degradation products of moxidectin

OH

OH

H3C

H3 C

CH3

CH3

H
O

C5 Protection

H3C
O

HO

CH3

H3C

H3 C

H
O

CH3

H3 C

H3C

HO

CH 3

H3 C

CH3

H
OH

Nemadectin

PG

C5 protected intermediate

C23 Oxidation
O
O

H3 C

H3C

H3C

CH3

CH3
H

O
CH3

H3C

H3 C

H
O

HO

H3C

C23 Oxime f ormation

HO

CH3

H3 C

H3C

CH3

H3C

CH3
H

PG

PG

C5 protected C23-keto intermediate

Or

C5 protected C23-methoxime
intermediate

C5 Deprotection

C5 Deprotection
O

H3C

H3C

CH3
H

H3C

CH3
H

O
H3C
CH3

H3C
H3C

H
O

HO

CH 3

H3C

C23 Oximation

H3C

HO

H 3C

CH3
H

OH

CH3
H
OH

C5 protected C23-keto intermediate

Moxidectin
Where PG is protecting group

Fig. 9 Schematic process for conversion of nemadectin into moxidectin [36]

impurities rather than DPs, with the exception of impurities

D and E, which are base-catalyzed DPs. Forced degradation
using acid produces two previously unknown impurities,
now characterized (using high-resolution MS and NMR
spectroscopy) as 3,4-epoxy-moxidectin and 23-ketonemadectin. An investigation into the degradation pathways
reveals the relationship of 23-keto-nemadectin to the manufacturing process used for MOX. 3,4-Epoxy-moxidectin

observed under acid hydrolysis is an acid-catalyzed oxidation product of MOX. The data gathered in this work will
provide crucial information on typical DPs of MOX and its
formulation under stress studies and long-term storage.
This will be helpful to develop stable formulations of
MOX and provide supporting data to new pharmacopeial
monograph for MOX finished product as well as chemical
reactivity of MOX.

A. Awasthi et al.
Acknowledgments This research work was supported by Technology
New Zealand (TechNZ). The authors would like to take this opportunity
to thank Mr. Robert Holmes and Ancare Scientific Ltd, New Zealand for
their continuous support. Also, special thanks to Prof. Saranjit Singh
(NIPER, India) for his guidance on characterization protocols.

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