Send Orders of Reprints at reprints@benthamscience.org
Current Pharmaceutical Analysis, 2012, 8, 415-430 415
Separation and Identification of Degradation Products in Abamectin
Formulation Using LC, LTQ FT-MS, H/D Exchange and NMR
Atul Awasthi
1
, Majid Razzak
2
, Raida Al-Kassas
1
, David R. Greenwood
3
, Joanne Harvey
4
and
Sanjay Garg
1,5*
1
School of Pharmacy, The University of Auckland, Private Bag 92019, Auckland, New Zealand
2
Ancare Scientific Ltd, Auckland, PO Box 36240, Northcote, Auckland 0748, New Zealand
3
School of Biological Sciences, The University of Auckland, Private Bag 92019, Auckland, New Zealand
4
School of Chemical and Physical Sciences, Victoria University of Wellington, PO Box 600, Wellington, New Zealand
5
School of Pharmacy and Medical Sciences, University of South Australia, PO Box 2471, Adelaide, Australia
Abstract: This paper describes the analysis of abamectin and the characterization of its degradation products in formula-
tion. A fast and selective reversed-phase HPLC was developed and validated for the quantitation of degradation products.
Two major degradation products observed in stress study samples were isolated and characterized using chromatography
and high resolution FT-MS. Abamectin and its degradation products presented similar masses at m/z 873.49848 Da, and
were further distinguished by MS
n
studies, H/D exchange studies,
1
H,
13
C and 2D NMR experiments. The interpretation of
analytical data positively identified unknown 1 as the stereoisomer 2-epi-abamectin and unknown 2 as the regioisomer
2,3-abamectin. The developed HPLC method was found to be precise, accurate and detector response was linear for the
analysis of known degradation products at the identification threshold.
Keywords: Abamectin, Degradation products, FT-MS, H/D exchange, HPLC, Impurity isolation, In-silico Fragmentation,
MS
n
, NMR, Structural and Stereoisomer.
1. INTRODUCTION
Abamectin [ABM] belongs to a group of compounds,
commonly known as the avermectins (AVMs) and is com-
mercially available as a mixture of two closely related ho-
mologs (see Fig. 1) ABM B1
a
(> 90%; M.W.: 873.08) and
ABM B1
b
(< 10%; M.W.: 859.05) [1, 2]. ABM is widely
used as an anthelmintic agent and currently licensed for the
treatment of a broad spectrum of endo- and ecto-parasites of
farm animals (e.g. gastrointestinal roundworms, lungworms,
lice, grubs and mange mites) [3-6].
The AVMs molecular structure consists of a 16-
membered macrocyclic lactone (ML) ring containing a spi-
roketal group and a benzofuran ring attached to a modified
disaccharide via glycosidic linkage [7]. These are highly
lipophilic substances and dissolve in most organic solvents.
ABM is isolated from the mixture of AVMs obtained from
the fermentation of soil bacterium Streptomyces avermetilis
[8]. ABM can be chemically modified to obtain other well-
known derivatives like ivermectin (22,23-dihydroaver-
mectin), eprinomectin (4-deoxy-4-epi-acetylamino-aver-
mecctin) and other derivatives [9, 10].
*Address correspondence to this author at the School of Pharmacy and
Medical Sciences, University of South Australia, PO Box 2471, Adelaide,
Australia; Tel: +61 8 8302 1575, Fax: +61 8 8302 2389;
E-mail: sanjay.garg@unisa.edu.au
Fig. (1). Chemical structure and molecular formula of ABM (B1a
& B1b).
Officially, ABM is not monographed in any compen-
dium; however there are some reports published on the High
Performance Liquid Chromatography (HPLC) analysis of
ABM and its derivatives [2]. The majority of studies have
been focused on environmental, food, milk and other matri-
ces using different extraction and detection methods [11-15].
The analysis of ABM has also been reported in the presence
of a few known degradents/metabolites; Pivnichny et al.
O
O
R
CH
3
O O
CH
3
OH
O
H
3
C
O
HO
CH
3
H
H
H
H
H
O
O
O
H
3
C
OCH
3
H
3
C
OCH
3
HO
H
3
C
Avermectin B1a: R = C2H5
Avermectin B1b: R = CH3
1875-676X/12 $58.00+.00 © 2012 Bentham Science Publishers