1340 SCHENCK & LAGMAN: JOURNAL OF AOAC INTERNATIONAL VOL 82, NO.
6, 1999
DRUGS, COSMETICS, FORENSIC SCIENCES
Multiresidue Determination of Abamectin, Doramectin,
Ivermectin, and Moxidectin in Milk Using Liquid
Chromatography and Fluorescence Detection
SCHENCK & LAGMAN: JOURNAL OF AOAC INTERNATIONAL VOL 82, NO. 6, 1999
FRANK J. SCHENCK1
U.S. Food and Drug Administration, Baltimore District Laboratory, 900 Madison Ave, Baltimore, MD 21201
LAWRENCE H. LAGMAN
U.S. Department of Agriculture, Agricultural Marketing Service, Eastern Laboratory, 645 Cox Rd, Gastonia, NC 28054
Abamectin, doramectin, ivermectin, and moxidectin
are macrocyclic lactones derived from soil dwell-
ing actinomycetes, and are very effective against
nematode, insect, and arthropod infestations.
These compounds, known as endectocides, have
been approved for use in beef cattle in the United
States; however, they are currently not approved
for use in dairy cattle. Abamectin, doramectin,
ivermectin, and moxidectin residues were isolated
from milk by a series of liquid–liquid extraction
steps, derivatized with trifluoroacetic anhydride,
and determined by liquid chromatography with flu-
orescence detection. Recovery studies were per-
formed in 2 laboratories. Recoveries of >80%
(1–30 ng/mL) were achieved for all 4 compounds.
he avermectins and milbemycins are closely related
T 16-membered macrocyclic lactones derived from
soil-dwelling actinomycetes of the genus Streptomyces.
They are extremely potent against nematode and arthropod
species as well as acarine and insect plant pathogens. Their
use in the treatment of endoparasitic infections and
ectoparasitic infestations gave rise to the name endectocide.
The primary structural difference between the groups is that
the avermectins have a disaccharide moiety attached to the
13-position of the macrocyclic ring, whereas the milbemycins
do not (1).
Eight avermectin compounds have been isolated by ex-
tracting the mycelia of S. avermitilis. These consist of 4 major
“a” compounds and 4 minor (5–10%) “b” homologues, desig-
nated A1a through B2b (2). Abamectin, a mixture of >80%
avermectin B1a and <20% avermectin B1b, was first intro-
duced as an agricultural pesticide and later as an animal
endectocide (3–5). Ivermectin, a mixture of >80%
22,23-dihydroavermectin B1a, and <20%
22,23-dihydroavermectin B1b, is produced by the selective hy-
Received January 29, 1999. Accepted by JM June 29, 1999.
1 column (Rainin Instrument Co., Woburn, MA). Operating
Current address: U.S. Food and Drug Administration, 60 Eighth St,
NE, Atlanta, GA 30309.
drogenation of avermectin B1 (6). Doramectin is produced by
mutational biosynthesis, feeding cyclohexanecarboxylic acid
to a mutant strain of S. avermitilis (7, 8). Moxidectin is ob-
tained by the chemical modification of the milbemycin,
nemadectin, which is produced by S. cyanogriseus (9). (See
Figure 1 for chemical structures.) An excellent review of the
avermectins and milbemycins has been presented by McKel-
lar and Benchaoui (10).
The avermectins and milbemycins are highly lipophilic
and are stored in fat tissue, which acts as a reservoir, contribut-
ing to their long-term persistence in the body (11). Ivermectin
and moxidectin have been found in the milk of lactating ani-
mals many weeks after dosing (12–14). None of the
4 endectocides discussed above are approved for use in lactat-
ing dairy cattle in the United States (15).
Methods for the determination of ivermectin in milk have
been reported in the literature (16–21). Lagman (22) demon-
strated that abamectin, doramectin, and moxidectin could be
extracted from milk by using a method first developed for
ivermectin in milk. A multiresidue liquid chromato-
graphic (LC) method for the determination of abamectin,
doramectin, ivermectin, and moxidectin in milk is described
here.
METHOD
Apparatus
(a) Centrifuge.—Equipped with rotors for handling 15
and 50 mL centrifuge tubes.
(b) Centrifuge tubes.—Glass, 15 mL graduated and
50 mL, with polyethylene stoppers.
(c) Ultrasonic bath.—Bransonic Model 2210 (Branson
Ultrasonic Corp., Danbury, CT) or equivalent.
(d) Liquid chromatograph.—Model 650-15 fluorescence
spectrophotometer, Series 410 LC pump, Model
ISS-100 autoinjector (Perkin-Elmer Corp., Norwalk, CT);
4.6 × 150 mm Chromegabond MC18 LC column (ES Indus-
tries, W. Berlin, NJ) with Brownlee Newguard RP-18 guard
conditions: excitation wavelength, 364 nm; emission wave-
 SCHENCK & LAGMAN: JOURNAL OF AOAC INTERNATIONAL VOL 82, NO. 6, 1999 1341
Figure 1. Chemical structures of abamectin, doramectin, ivermectin, and moxidectin.
length, 475 nm; injection volume, 100 µL; column tempera-
ture, 30°C; solvent flow rate, 0.7 mL/min.
Reagents
(a) Solvents.—LC grade acetonitrile, ethyl acetate, hexane,
2,2,4-trimethylpentane (isooctane), and tetrahydrofuran (THF);
USP ethanol; ACS reagent grade ammonium hydroxide.
(b) Water.—Deionized (Milli-Q Water System, Waters
Corp., Milford, MA) or equivalent.
(c) LC mobile phase.—Acetonitrile–THF–water
(90 + 6 + 4; v/v/v). Add 100 mL acetonitrile, 40 mL water,
and 60 mL THF to 1.0 L volumetric flask. Add acetonitrile to
volume and mix.
(d) Silylation reagent.—Sylon-CT (Supelco, Inc.,
Bellefonte, PA).
(e) N-methylimidazole.—99% pure (Aldrich Chemical
Co., Milwaukee, WI).
(f) Trifluoroacetic anhydride (TFAA).—Pierce, Rockford, IL.
(g) Derivatizing reagent.—Mix 4 mL acetonitrile with
2 mL TFAA immediately before use.
(h) Milk.—Raw milk was obtained from the U.S. Depart-
ment of Agriculture, Beltsville, MD, and stored at –70ºC. Ho-
mogenized, grade A milk was obtained from local grocery
stores and stored at 4ºC.
Standards
(a) Abamectin reference standard.—0.819% (w/w)
abamectin B1a and 0.054% (w/w) abamectin B1b in glycerol
formal (Merial Ltd., Three Bridges, NJ).
(b) Doramectin reference standard.—Pfizer Research
Laboratories, Groton, CT.
(c) Ivermectin reference standard.—1.38% (w/w)
ivermectin B1a and 0.21% (w/w) ivermectin B1b in glycerol
formal (Merck Sharpe and Dohme Research Laboratories,
Rahway, NJ).
(d) Moxidectin reference standard.—Agricultural Re-
search Division, American Cyanimid Corp., Princeton, NJ.
Preparation and Handling of Silylated Tubes
Graduated centrifuge tubes (15 mL) were thoroughly
cleaned, silylated with Sylon CT for 30 min, rinsed with tolu-
ene and methanol, soaked with methanol for 30 min, and
rinsed with acetone. The tubes may be used for 2 months be-
fore the silylation procedure is repeated.
Silylated Tube Cleaning
After use the tubes are soaked for several hours in methy-
lene chloride and then in detergent solution. They are rinsed
with hot water, distilled water, and acetone. They must be
1342 SCHENCK & LAGMAN: JOURNAL OF AOAC INTERNATIONAL VOL 82, NO. 6, 1999
cleaned by hand. If they are machine washed, the silylation
procedure must be repeated.
Standard Solutions
(a) Stock standard solutions.—Solutions (100 µg/mL) of
abamectin, doramectin, ivermectin, and moxidectin in
acetonitrile were prepared and stored in actinic glassware at
–20°C.
(b) Mixed working standard solution (1.0 mg/mL).—From
each of the stock standard solutions 1.0 mL was added to a
100 mL actinic glass volumetric flask. Acetonitrile was added
to volume. The solution was stored at –20°C.
Sample Extraction
Add 10.0 mL milk to 50 mL centrifuge tube. For recovery
studies, add appropriate amount of working standard to milk,
Vortex mix, and wait 15 min. Add 2.5 mL concentrated am-
monium hydroxide, and mix. Add 10 mL ethanol, 10 mL ethyl
acetate, and 10 mL isooctane sequentially, shaking mixture
30, 30, and 20 s after each respective addition. Centrifuge
sample 4 min to separate liquid phases; transfer upper layer to
second 50 mL centrifuge tube with pipette. Add additional
10 mL ethyl acetate and 10 mL isooctane sequentially to first
tube, shaking mixture as above; repeat centrifugation. Com-
bine top layers in second tube, and evaporate under stream of
nitrogen at 70°C. Dissolve oily residue by adding ca 4 mL
hexane, sonicating, and Vortex mixing. Quantitatively trans-
Figure 2. Typical chromatograms of (A) control raw milk and (B) control raw milk fortified with 2.0 ng/mL abamectin,
doramectin, ivermectin, and moxidectin. Peak identities are (1) moxidectin, (2) abamectin B1b, (3) abamectin B1a,
(4) doramectin, (5) ivermectin B1b, (6) ivermectin B1a.
fer hexane to graduated 15 mL centrifuge tube, evaporate to
<3 mL under stream of nitrogen, and adjust volume to 3.0 mL
with hexane. Add 4.0 mL acetonitrile, shake 1 min, centrifuge
4 min, transfer lower acetonitrile phase to 15 mL silylated cen-
trifuge tube. Repeat acetonitrile extraction. Shake combined
acetonitrile extracts with 1 mL hexane, centrifuge 4 min, and
discard hexane. Evaporate acetonitrile to dryness under nitro-
gen at 70°C.
Derivatization
Less than 0.050 mL of residue should remain. (Note: The
presence of moisture, methanol, or excess milk matrix resi-
due, caused by incomplete removal of the hexane layer during
sample preparation, can interfere with the derivatization reac-
tion.) Reconstitute sample extract in 2.0 mL acetonitrile. Pre-
pare series of standards for standard curve by adding mixed
working standard to 15 mL silylated centrifuge tubes. (Note: If
methanolic working standards are used, they must first be
evaporated to dryness.) Add acetonitrile to the 2 mL mark.
Add 0.2 mL N-methylimidazole and Vortex mix.
Add 0.6 mL freshly prepared derivatization reagent to
silylated tubes containing samples and standards. Stopper
tubes, Vortex briefly, and store in the dark. After 15 min, di-
lute solutions to 5.0 mL with acetonitrile, Vortex mix, and
store shielded from direct light. Inject 100 µL of each sample
and standard into LC system. Measure peak heights at reten-
 SCHENCK & LAGMAN: JOURNAL OF AOAC INTERNATIONAL VOL 82, NO. 6, 1999 1343

Table 1. Recovery of fortified abamectin, doramectin, ivermectin, and moxidectin residues from milk
Recovery, %a

Fortification, ng/mL Abamectinb Doramectin Ivermectinb Moxidectin

Homogenized milk

3 90.9 (9.4) 95.5 (8.5) 90.7 (13.6) 104.7 (23.7)

6 91.1 (17.4) 91.6 (20.3) 93.7 (21.2) 100.1 (15.3)
15 84.6 (10.7) 87.1 (12.1) 86.9 (14.5) 91.1 (11.9)
30 83.5 (3.1) 84.1 (4.4) 83.7 (7.0) 89.8 (7.2)
Raw milk

1 109.9 (3.3) 108.7 (5.2) 111.3 (3.6) 113.8 (17.4)

2 99.7 (0.7) 101.5 (1.7) 102.6 (1.7) 102.3 (2.4)
5 99.7 (2.9) 98.2 (2.7) 99.8 (2.8) 98.5 (2.4)
10 97.6 (2.4) 99.4 (2.0) 101.9 (0.6) 98.4 (1.8)
15 98.7 (2.3) 100.6 (1.8) 99.5 (1.5) 100.3 (0.6)
30 100.8 (2.3) 103.0 (2.1) 102.4 (2.1) 103.1 (2.3)

a
n = 4; values in parentheses are coefficients of variation.
b
Recoveries are for abamectin B1a and ivermectin B1a.

tion times of abamectin B1a, doramectin, ivermectin B1a, and
moxidectin derivatives as indicated by standards.
Generation of Standard Curves and Calculations
For each of the 4 endectocides, calculate slope, a, and inter-
cept, b, of a standard curve by linear regression analysis of stan-
dards based on peak height of the endectocide derivative peaks,
using y = ax + b, where y = peak height and x = concentration
of standard in ng/mL. Using peak heights of endectocide deriv-
atives in the milk sample, calculate concentration injected into
the LC system using the following equation:
Analyte injected into LC system, ng/mL =
(peak height – b)/a
Calculate concentration of analyte in milk sample as fol-
lows:
Milk sample, ng/mL =
(analyte injected, ng/mL) × (V1/V2 ) or
Milk sample, ng/mL =
(analyte injected, ng/mL) × 0.5
where V1 = final volume of derivatized extract (5.0 mL) and
V2 = volume of milk extracted (10 mL).
Results and Discussion
The liquid chromatographic method with fluorescence de-
tection is both specific and sensitive. The extraction and
cleanup is similar to the method presently used by the U.S.
Food and Drug Administration (FDA) for the determination
of ivermectin in milk (18). The 4 lipophilic endectocides
(abamectin, doramectin, ivermectin and moxidectin) were ex-
tracted from milk along with milk fat, and separated from fat
by acetonitrile/hexane partitioning.
Although all 4 of the endectocides have UV chromophores,
the presence of coextracted milk matrix components pre-
cludes using UV detection at the low levels. Tway et al. (23)
first reported that fluorescent derivatives of ivermectin can be
formed by reacting it with acetic anhydride (AA) at 95°C in
the presence of a base catalyst, N-methylimidazole (NMIM).
The AA–NMIM derivatization reaction results in many re-
agent by-products; thus further cleanup of the derivatized
standards and samples is required. De Montigny et al. (24) re-
ported that when trifluoroacetic anhydride (TFAA) was sub-
stituted for AA, the derivatization of avermectins took place in
<30 s at 25°C. Because substantially fewer reagent
by-products were formed by the TFAA derivatization, fur-
ther cleanup of the derivatized standards and samples was
not required.
The TFAA derivatives of the 4 endectocides are very hy-
drophobic. Liquid chromatographic separation is usually
achieved by reversed-phase octadecylsilica (C18) or
octylsilica (C8) columns with mobile phases containing a very
high percentage of organic solvent. Typically, mixtures of
methanol–water (95 + 5) or acetonitrile–water (97 + 3), with
flow rates ranging from 1.2 to 2.0 mL/min, are used. Even
with these high percentages of organic modifier and relatively
high flow rates, retention times of 12–15 min for ivermectin
are common. We achieved similar retention times using a
0.7 mL/min flow rate with an acetonitrile–THF–water
(90 + 6 + 4) mobile phase. The lower flow rate resulted in sig-
nificantly better chromatography, with a greater than 2-fold
1344 SCHENCK & LAGMAN: JOURNAL OF AOAC INTERNATIONAL VOL 82, NO. 6, 1999
increase in peak heights compared to acetonitrile–water or
methanol–water mobile phases at higher flow rates. Typi-
cal chromatograms of fortified and control milks are (4)
shown in Figure 2.
Recovery studies of the 4 endectocides were performed in (5)
2 different laboratories. Recovery studies using homogenized (6)
milk were first performed at one laboratory using an
acetonitrile–water (97 + 3) mobile phase. Recovery studies
(7)
were later performed on raw milk, using the improved
acetonitrile–THF–water (90 + 6 + 4) mobile phase at the sec-
ond laboratory. Table 1 shows that recoveries ranging from 83
(8)
to 114% at 1–30 ng/mL levels were obtained for the
4 endectocides by the 2 laboratories. The CVs were lower for
raw milk than for homogenized milk because of improved (9)
chromatography and the use of the acetonitrile–THF–water
mobile phase with raw milk samples. The limits of detection
(S/N = 3) for the 4 endectocides were ca 0.3 ng/mL when this (10)
mobile phase was used.
Two potential problems with the TFAA derivatization (11)
have been reported. If any water is present in the injection ma-
trix, excess TFAA may be hydrolyzed to trifluoroacetic acid, (12)
resulting in breakdown of the derivative (25, 26). Therefore,
the derivatized standards and samples were diluted with (13)
acetonitrile and not with mobile phase which contains water.
De Groodt et al. (27) reported that TFAA derivatives of (14)
ivermectin are unstable in the presence of light and will un-
dergo 50% degradation in 3 h when exposed to daylight. Al- (15)
though some degradation of doramectin TFAA derivatives
was noted after 3 h when exposed to laboratory fluorescent (16)
lighting, no degradation of the TFAA derivatives of the
4 endectocides was observed over 6 h when the derivatives (17)
were shielded from direct light.
(18)
(19)
Acknowledgments
(20)
Funding for the work performed at the Agricultural Mar-
(21)
keting Service Laboratory was provided by the USDA Pesti-
cide Data Program. (22)
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