ANALYST, SEPTEMBER 1989, VOL.
114
Determination of Nitroxynil Residues in Tissues Using
High-performance Liquid Chromatography - Thermospray Mass
Spectrometry
W. John Blanchflower and D. Glenn Kennedy
Department of Agriculture, Veterinary Research Laboratories, Stormont, Belfast BT4 3SD, UK
A method is described for the determination of nitroxynil residues in muscle, liver and kidney. The samples
were extracted into diethyl ether and cleaned-up using a simple liquid - liquid extraction step. Any nitroxynil
present was separated from interfering compounds by high-performance liquid chromatography and
detected using thermospray mass spectrometry. The assay is specific and sensitive, with a detection limit of
2 ng 9-1 in tissues.
Keywords: Nitroxynil; residue; high-performance liquid chromatography; thermospray mass spectrometry
Nitroxynil (4-hydroxy-3-iodo-5-nitrobenzonitrile)is an
anthelmintic mainly used to control liver fluke (Fasciola
hepatica) in sheep and cattle.1 It is marketed as an aqueous
solution of the N-ethylglucamine salt under the name "Tro-
dax." As residues of the drug have been detected in tissues of
animals up to 90 d after treatment,2 adequate withdrawal
times must be observed before animals are slaughtered for
human consumption. This has led t o the necessity for the
development of methods to monitor residual levels of nitroxy-
nil in meat products to ensure that they fulfil the relevant
government tolerance limits.
Most of the results reported for nitroxynil have been
obtained using a polarographic method3 and, more recently, a
gas chromatographic method has been described for deter-
mining residue levels in milk.4 However, for statutory residue
testing purposes, mass spectrometric methods should be used
if possible to ensure adequate specificity.
This paper describes the development of a high-perfor-
mance liquid chromatographic - thermospray mass spectro-
metric (HPLC - MS) method for the detection and quantifica-
tion of nitroxynil residues in the tissues of slaughtered
animals.
Experimental
Apparatus
The HPLC system consisted of a Merck-Hitachi Model
L-6000 pump (BDH, Romford, Essex, UK), a Rheodyne
Model 7125 injector fitted with a 2 0 4 sample loop (BDH)
and a LiChrosorb RP-18, 250 X 4 mm i.d. reversed-phase
cartridge with holder (BDH).
The HPLC - MS system was a Vestec Model 201A thermo-
spray instrument (Vestec, Houston, TX, USA) complete with
a Technivent workstation. The instrument was operated in the
negative ion chemical ionisation mode (CI) using filament-
initiated ionisation with an electron beam current of 250 FA.
The electron multiplier voltage was 2400 V. The temperatures
of the vaporiser, block, tip heater and lens assembly were set
at 180, 250, 280 and 150"C, respectively. The tuning para-
meters were checked weekly according to the manufacturer's
instructions using a 50 mg 1-1 solution of polyethylene glycol
300 in 50% acetonitrile containing 0.1 M ammonium acetate
solution. The instrument was used either in the full-scan mode
to collect spectra or in the selected ion monitoring mode
(SIM) for maximum sensitivity when analysing samples. For
the former, a dwell time of 0.1 ms was used and for the latter
the dwell time was 100 ms. In each instance the sweep window
was set at 0.5 a.m.u.
1013
Reagents
Acetonitrile was of HPLC grade; all other reagents were of
AnalaR grade. Nitroxynil was obtained as the N-ethylglucam-
ine salt from May & Baker, Dagenham, Essex, UK. A stock
standard (1 -755 mg ml-1 of the salt, equivalent to 1mg ml-1 of
nitroxynil) was prepared in methanol and was stable for 1
month if stored at 4°C. A dilute standard (100 ng ml-1 of
nitroxynil) was prepared daily by dilution of the stock
standard with the mobile phase. The mobile phase consisted of
a mixture of 300 ml of acetonitrile and 700 ml of 0.1 M
ammonium acetate solution. This solution was de-gassed and
filtered under vacuum through a 0.45-pm filter using a
Millipore - Waters solvent filtration system (Millipore -
Waters, Harrow, Middlesex, UK). The mobile phase was
pumped at a flow-rate of 1 ml min-1.
Method
Cut the tissue samples into small cubes and store at -20°C
until frozen or until required. Place the frozen cubes in a
domestic food blender and pulverise until the tissue forms a
fine powder. Weigh 2-g portions into 110 x 25 mm centrifuge
tubes fitted with ground-glass necks. Add 15 ml of 0.4 M
disodium hydrogen orthophosphate solution and homogenise
for 1 min using a Silverson homogeniser. Add 2 ml of
concentrated hydrochloric acid and cool the tubes by allowing
them to stand in cold water for a few minutes. Add 8 ml of
diethyl ether, stopper the tubes and shake them vigorously by
hand for 30 s. Centrifuge at 10°C and 2000 g for 10 min.
Remove the upper diethyl ether layer using a Pasteur pipette
and transfer it into 100 x 16 mm centrifuge tubes fitted with
ground-glass necks. Reduce the volume of the diethyl ether to
a few millilitres under nitrogen at 40°C in a fume cupboard.
Re-extract the homogenates once with further 5-ml aliquots of
diethyl ether and combine these with the first extracts. Again
reduce the volume of the diethyl ether to about 4 ml under
nitrogen. Add 3 ml of 0.4 M disodium hydrogen orthophos-
phate solution, stopper the tubes and shake them vigorously
by hand for 30 s. Centrifuge at 10°C and 2000 g for 5 min.
Remove the upper diethyl ether layer and discard, taking care
not to remove any of the aqueous layer. Add 0.4 ml of
concentrated hydrochloric acid and cool the tubes by allowing
them to stand in cold water for a few minutes. Add 2 ml of
diethyl ether, stopper the tubes and shake them vigorously by
hand for 20 s. Centrifuge at 10°C and 2000 g as before.
Transfer the upper diethyl ether layer into 70 x 12 mm tubes,
again fitted with ground-glass necks. Re-extract the acidified
orthophosphate solutions once with further 1-ml aliquots of
diethyl ether and combine these with the first extracts.
1014 ANALYST, SEPTEMBER 1989, VOL. 114

Evaporate to dryness at 40°C under nitrogen in a fume
-upboard. Dissolve the residue in 100 pl of acetonitrile by
eating at 60 "C for a few minutes and mix. Add 200 pl of 0.1 M
mmonium acetate solution and mix. Inject 20-11 aliquots into
ie HPLC - MS system using the Rheodyne injector. Collect
ie peak data using the workstation and record the areas of the
eaks. Compare these with peak areas for 20-pl aliquots of a
30 ng ml-1 nitroxynil standard similarly injected.
Results
'he HPLC-MS CI spectrum of nitroxynil obtained by
ijecting 20 pl of a 10 pg ml-1 standard into the system and
dlecting all the spectral data is shown in Fig. 1. Two main
ms were observed, at rn/z 273 and m/z 290 (the molecular
m). For optimum sensitivity when analysing tissue samples,
ither ion could be monitored by STM. Alternatively, if more
48131 .
40000 - ?H
30000 - of a blank tissue sample with 10 and SO ng 8-1 of nitroxynil and
20000 - procedure. The results for the mean (standard deviation) and
10000 -
0 ' I I
I I I
II, I 1
definitive proof of the presence of nitroxynil residues in the
tissue samples is required, both ions can be monitored and the
ion ratio measurements compared.
Typical computer outputs for the ion chromatograms at m/z
290 for a standard, a blank tissue extract and a tissue extract
from a muscle sample spiked with 10 ng g-1 of nitroxynil are
shown in Fig. 2. Nitroxynil elutes at 4.8 min.
The linearity of the assay was checked by spiking duplicate
aliquots of a blank tissue sample with 10, SO, 150,250,400 and
SO0 ng g-1 of nitroxynil and carrying them through the
procedure. The results are shown in Fig. 3 and it can be seen
that the assay is linear up to at least 500 ng g- I of nitroxynil in
tissues. For samples containing more than SO ng g-1 of
nitroxynil, the final extracts were diluted with an appropriate
volume of the mobile phase before injection to prevent
saturation of the electron multiplier signal.
The recovery of nitroxynil in the assay was determined using
the results from the linearity test. The measured peak areas
were compared with those of a 10 ng ml-1 standard and the
amount of nitroxynil recovered was determined. The results
are shown in Table 1; recoveries ranged from 71 to 95%.
The precision of the assay was checked by spiking aliquots
then analysing each aliquot five times using the described
coefficient of variation were 8.42 (1.03) ng g-1 and 12.2% for
the 10 ng g-1 samples and 44.4 (2.53) ng g-1 and 5.7% for the
SO ng g-1 samples.

'
g. 1. Full-scan negative-ion CI spectrum from mlz 250 to mlz 320
the nitroxynil peak at 4.8 min; 20 yl of a 10 yg ml-1 standard were
jecred. The molecular structure of nitroxynil is also shown 67
5000 K
.E 4000
3
2
.- 3000
e
16000 m' 2000
2
14000

12 000

10 000
0 - I I I I I
8000
0 100 200 300 400 500
6000 Amount of nitroxynilhg g-1

4000 Fig. 3. Linear regression of a series of extracts from muscle samples

spiked with 1&500 ng g-I of nitroxynil and carried through the assay;
a, injection volume, 20 yl. Extracts from samples spiked with >50 ng g-1
52
of nitroxynil were diluted with the mobile phase before injection to
prevent saturation of the electron multiplier signal

.-w
c

2 3000
a,
[r
' I I I I 1
Table 1. Recovery of nitroxynil added to tissue samples

Added/ng g-' Found& g- Recovery, YO

10 8.4 84
- ('' 50 44.5 89
13 000
150 106.2 71
250 236.7 95
11000 - 400 284.9 71
500 413.6 83
9000 -
Table 2. Nitroxynil levels in muscle and kidney from treated cattle

Nitroxynil/ng 8-1

3000 ' 1
1
2
I
3 4
1 I

5
1
6
I
7
Sample
1
2
Muscle
300
130
Kidney
520
160
Run t i m e h i n
3 540 780
g. 2. Ion chromatograms at m/z 290 of a 2 0 4 injection of ( u ) a 100 4 540 790
; ml-I nitroxynil standard solution (nitroxynil elutes at 4.8 min); (6) 5 360 -
blank muscle extract; and ( c ) a tissue extract from a muscle sample 6 510 -
iked with 10 ng g-I of nitroxynil
ANALYST. SEPTEMBER 1989, VOL. 114
The results for a batch of samples submitted to this
laboratory from an abattoir as part of a residue testing scheme
are shown in Table 2. It was believed the animals concerned
had been treated with the drug and submitted for slaughter
within the recommended withdrawal period of 28 d . The
values ranged from 130 to 540 ng g-1 for muscle and from 160
to 790 ng g-1 for kidney.
Discussion
High-performance liquid chromatography - mass spec-
trometry offers analytical laboratories a further instrumental
technique for the specific analysis of a wide range of chemical
compounds. Using dedicated HPLC - MS systems such as the
Vestec thermospray instrument described here, the analysis is
easier than using gas chromatography coupled with mass
selective detection. Derivatisation is generally not required
and clean-up steps are often relatively simple. There are two
main reasons for this: (1) the sensitivity of thermospray
H P L C - M S using Cl is compound dependent, with some
compounds such as nitroxynil giving good sensitivity whereas
others are relatively insensitive; and (2) little fragmentation of
the molecules takes place, with mainly the mass ion and a few
large fragment ions being produced. In the proposed method,
the only clean-up step required after the initial extraction is for
the removal of fats. This was achieved by extracting the
nitroxynil from diethyl ether into disodium hydrogen ortho-
phosphate solution, acidifying the extract and re-extracting
into diethyl ether. As can be seen from Fig. 2, no interfering
compounds are present in the ion chromatograms of the ion at
rnfz 290.
The Vestec HPLC - MS instrument used offers three modes
of ionisation: (1) pure thermospray; (2) filament-assisted CI;
1015
and (3) Townsend discharge assisted CT. For nitroxynil we
found that filamcnt-assisted CI gave the best sensitivity and
this form of ionisation was therefore used for the assay. The
instrument also allows the measurement of both positive and
negative ions, and operation in the negative-ion mode was
again found to offer the greater sensitivity for nitroxynil.
The assay was found to be linear up to at least 500 ng g-1 of
nitroxynil in tissues (Fig. 3) and recoveries averaged 82%
(Table 1). It was not, therefore, necessary to construct a
calibration graph for each batch of samples. A 100 ng ml-1
standard injected after each batch of five sample extracts was
found to be sufficient.
The proposed assay was used to confirm the presence of
nitroxynil residues in muscle, liver and kidney samples
submitted from abattoirs as part of our residue testing
programme. A batch of 15 samples can easily be analysed by
one operator in one working day and the results are more
specific than those obtained with previously reported
methods. The sensitivity is also higher, with a detection limit
of 2 ng 6-1 in tissues, compared with 100 ng g-1 when using the
polarographic method.3
References
1. Lucas. J. M . S . , Rr. Vet. J . . 1967, 123, 198.
2. Ekstrom, L. G., and Slania, P., Actu Ver. Scand., 1982,23,313.
3. Parnell, M. J., Pestic Sci., 1970. 1. 138.
4. Kazacos, M . , and Mok, V., Aust. J . Dairy Technol., 1986, 41,
82.
Paper 9i00835G
Received February 23rd, 1989
Accepted April 17th, 1989