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114 1013

Determination of Nitroxynil Residues in Tissues Using

High-performance Liquid Chromatography - Thermospray Mass
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 Reagents

anthelmintic mainly used to control liver fluke (Fasciola
Acetonitrile was of HPLC grade; all other reagents were of
hepatica) in sheep and cattle.1 It is marketed as an aqueous
AnalaR grade. Nitroxynil was obtained as the N-ethylglucam-
solution of the N-ethylglucamine salt under the name "Tro-
ine salt from May & Baker, Dagenham, Essex, UK. A stock
dax." As residues of the drug have been detected in tissues of standard (1 -755 mg ml-1 of the salt, equivalent to 1mg ml-1 of
animals up to 90 d after treatment,2 adequate withdrawal nitroxynil) was prepared in methanol and was stable for 1
times must be observed before animals are slaughtered for month if stored at 4°C. A dilute standard (100 ng ml-1 of
human consumption. This has led t o the necessity for the nitroxynil) was prepared daily by dilution of the stock
development of methods to monitor residual levels of nitroxy- standard with the mobile phase. The mobile phase consisted of
nil in meat products to ensure that they fulfil the relevant a mixture of 300 ml of acetonitrile and 700 ml of 0.1 M
government tolerance limits. ammonium acetate solution. This solution was de-gassed and
Most of the results reported for nitroxynil have been
filtered under vacuum through a 0.45-pm filter using a
obtained using a polarographic method3 and, more recently, a
Millipore - Waters solvent filtration system (Millipore -
gas chromatographic method has been described for deter-
Waters, Harrow, Middlesex, UK). The mobile phase was
mining residue levels in milk.4 However, for statutory residue
pumped at a flow-rate of 1 ml min-1.
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- Method
metric (HPLC - MS) method for the detection and quantifica- Cut the tissue samples into small cubes and store at -20°C
tion of nitroxynil residues in the tissues of slaughtered until frozen or until required. Place the frozen cubes in a
animals. 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
Experimental 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
The HPLC system consisted of a Merck-Hitachi Model diethyl ether, stopper the tubes and shake them vigorously by
L-6000 pump (BDH, Romford, Essex, UK), a Rheodyne hand for 30 s. Centrifuge at 10°C and 2000 g for 10 min.
Model 7125 injector fitted with a 2 0 4 sample loop (BDH) Remove the upper diethyl ether layer using a Pasteur pipette
and a LiChrosorb RP-18, 250 X 4 mm i.d. reversed-phase and transfer it into 100 x 16 mm centrifuge tubes fitted with
cartridge with holder (BDH). ground-glass necks. Reduce the volume of the diethyl ether to
The HPLC - MS system was a Vestec Model 201A thermo- a few millilitres under nitrogen at 40°C in a fume cupboard.
spray instrument (Vestec, Houston, TX, USA) complete with Re-extract the homogenates once with further 5-ml aliquots of
a Technivent workstation. The instrument was operated in the diethyl ether and combine these with the first extracts. Again
negative ion chemical ionisation mode (CI) using filament- reduce the volume of the diethyl ether to about 4 ml under
initiated ionisation with an electron beam current of 250 FA. nitrogen. Add 3 ml of 0.4 M disodium hydrogen orthophos-
The electron multiplier voltage was 2400 V. The temperatures phate solution, stopper the tubes and shake them vigorously
of the vaporiser, block, tip heater and lens assembly were set by hand for 30 s. Centrifuge at 10°C and 2000 g for 5 min.
at 180, 250, 280 and 150"C, respectively. The tuning para- Remove the upper diethyl ether layer and discard, taking care
meters were checked weekly according to the manufacturer's not to remove any of the aqueous layer. Add 0.4 ml of
instructions using a 50 mg 1-1 solution of polyethylene glycol concentrated hydrochloric acid and cool the tubes by allowing
300 in 50% acetonitrile containing 0.1 M ammonium acetate them to stand in cold water for a few minutes. Add 2 ml of
solution. The instrument was used either in the full-scan mode diethyl ether, stopper the tubes and shake them vigorously by
to collect spectra or in the selected ion monitoring mode hand for 20 s. Centrifuge at 10°C and 2000 g as before.
(SIM) for maximum sensitivity when analysing samples. For Transfer the upper diethyl ether layer into 70 x 12 mm tubes,
the former, a dwell time of 0.1 ms was used and for the latter again fitted with ground-glass necks. Re-extract the acidified
the dwell time was 100 ms. In each instance the sweep window orthophosphate solutions once with further 1-ml aliquots of
was set at 0.5 a.m.u. 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 definitive proof of the presence of nitroxynil residues in the
-upboard. Dissolve the residue in 100 pl of acetonitrile by tissue samples is required, both ions can be monitored and the
eating at 60 "C for a few minutes and mix. Add 200 pl of 0.1 M ion ratio measurements compared.
mmonium acetate solution and mix. Inject 20-11 aliquots into Typical computer outputs for the ion chromatograms at m/z
ie HPLC - MS system using the Rheodyne injector. Collect 290 for a standard, a blank tissue extract and a tissue extract
ie peak data using the workstation and record the areas of the from a muscle sample spiked with 10 ng g-1 of nitroxynil are
eaks. Compare these with peak areas for 20-pl aliquots of a shown in Fig. 2. Nitroxynil elutes at 4.8 min.
30 ng ml-1 nitroxynil standard similarly injected. 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
Results 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
'he HPLC-MS CI spectrum of nitroxynil obtained by
tissues. For samples containing more than SO ng g-1 of
ijecting 20 pl of a 10 pg ml-1 standard into the system and
nitroxynil, the final extracts were diluted with an appropriate
dlecting all the spectral data is shown in Fig. 1. Two main
volume of the mobile phase before injection to prevent
ms were observed, at rn/z 273 and m/z 290 (the molecular
saturation of the electron multiplier signal.
m). For optimum sensitivity when analysing tissue samples,
The recovery of nitroxynil in the assay was determined using
ither ion could be monitored by STM. Alternatively, if more
the results from the linearity test. The measured peak areas
were compared with those of a 10 ng ml-1 standard and the
48131 . amount of nitroxynil recovered was determined. The results
are shown in Table 1; recoveries ranged from 71 to 95%.
40000 - ?H The precision of the assay was checked by spiking aliquots
30000 - of a blank tissue sample with 10 and SO ng 8-1 of nitroxynil and
then analysing each aliquot five times using the described
20000 - procedure. The results for the mean (standard deviation) and
coefficient of variation were 8.42 (1.03) ng g-1 and 12.2% for
10000 -
the 10 ng g-1 samples and 44.4 (2.53) ng g-1 and 5.7% for the
0 ' I I
II, I 1 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
.- 3000
16000 m' 2000

12 000

10 000
0 - I I I I I
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
of nitroxynil were diluted with the mobile phase before injection to
prevent saturation of the electron multiplier signal


2 3000
' 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
3 4
1 I

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 1015

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