The Determination of Nitroimidazole Residues in Fish and Fish Products.pdf

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Analytica Chimica Acta 586 (2007) 394398

Confirmation of four nitroimidazoles in porcine liver by liquid

chromatographytandem mass spectrometry

X. Xia, X. Li, S. Zhang, S. Ding, H. Jiang, J. Shen

China Agricultural University, College of Veterinary Medicine, Department of Pharmacology and Toxicology, Beijing 100094, ChinaReceived 20 July 2006; received in revised form 30 August 2006; accepted 27 September 2006

Available online 10 October 2006

Abstract

A sensitive and reliable multiresidue method is described for analysis of ronidazole, metronidazole, dimetridazole and the common metabolite

of ronidazole and dimetridazole, 2-hydroxymethyl-1-methyl-5-nitroimidazole in swine liver. The sample preparation procedure was based on

liquidliquid extraction and mixed mode cation exchange/reverse phase solid-phase extraction. The compounds of interest were determined by

reverse phase gradient liquid chromatography separation and tandem mass spectrometry (MS/MS) in the multiple reaction monitoring (MRM)

mode. The limits of confirmation were 0.10.5 g kg1 for the analytes.

2006 Elsevier B.V. All rights reserved.

Keywords: Nitroimidazole; Liquid chromatographytandem mass spectrometry; Liver

1. Introduction

5-Nitroimidazoles area group of veterinary drugs used forthe

treatment of histomoniasis and coccidiosis in poultry and hem-

orrhagic enteritis in pigs. For their potentially harmful effects

on human health[1,2], nitroimidazoles have been banned from

use in food producing animals within the European Union[3],

the U.S.[4]and other countries including China[5].

Different methods have been developed for analysis of

nitroimidazoles and their metabolites in various tissues (mus-cle, liver, kidney and retina) as well as plasma, serum, egg,

faeces and water [621]. These methods ranged from less

selective methods such as gas chromatography coupled with

nitrogen and phosphorus detection (GC-NPD), liquid chro-

matography with ultra-violet detection and enzyme-linked

immunosorbent assay (ELISA) to confirmation methods includ-

ing gas chromatographymass spectrometry (GCMS) and liq-

uid chromatographymass spectrometry (LCMS). Polzer and

Gowik[12]described a multiresidue negative chemical ioniza-

tion (NCI) GCMS method for the confirmation of nitroimida-

zoles and their metabolites in turkey and swine muscle. The

same GCMS technique was applied by Ho et al. [14] for

Corresponding author. Tel.: +86 10 6273 2803; fax: +86 10 6273 1032.

E-mail address:[email protected](J. Shen).

the determination of dimetridazole (DMZ) and metronidazole

(MNZ) in a variety of tissue matrices. The main drawback of

the GCMS method is the derivatization step: the derivatization

products of ronidazole (RNZ) and 2-hydroxymethyl-1-methyl-

5-nitroimidazole (HMMNI) are identical.

As an alternative LCMSmethods have beendeveloped. Can-

navan and Kennedy [15] used LC with thermospray MS for

the determination of DMZ in poultry muscle, liver and eggs.

Sams et al. [16] described a LC-APCIMS method for the deter-

mination of DMZ, RNZ and HMMNI in poultry muscle andeggs. The limit of detection was 0.1g kg1 for DMZ and RNZ

and 0.5g kg1 for HMMNI in the single ion mode (SIM). A

LCMSMS method was presented by Daeseleire et al. [18]

for rapid confirmation of RNZ, MNZ and DMZ in eggs. The

method employed a relatively simple sample preparation with

method detection down to 0.5g kg1 for all three compounds.

Hurtaud-Pessel et al.[17]developed a LCMS method for the

analysis of nitroimidazole residues in poultrymeats, using RNZ-

d3 as internal standard. Similarly, Hormazabal and Yndestad

[19] determined DMZ, MNZ and RNZ in meat by LCMS using

external calibration.

We have previously demonstrated the usefulness of LC withelectrospray tandem mass spectrometry for the analysis of

nitroimidazoles in muscle and eggs at trace levels[22]. In the

current study, we use LCMSMS detection to simultaneously

confirm the presence of four nitroimidazoles residues in swine

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X. Xia et al. / Analytica Chimica Acta 586 (2007) 394398 395

liver at 0.10.5g kg1 level, which is appropriate for mon-

itoring illegal use of selected banned compounds in livestock

production.

2. Experimental

2.1. Chemicals and materials

HPLC grade methanol, acetonitrile, ethyl acetate and hex-

ane were purchased from Dima Technology Inc. (Muskegon,

MI). Hydrochloric acid and ammonia were provided by Bei-

jing Chemical Co. (Beijing, China). Water was purified using

a Milli-Q Synthesis system from Millipore (Bedford, MA).

The analytical standards RNZ, MNZ and DMZ were obtained

from Sigma (St. Louis, MO). HMMNI was from veteri-

nary drug safety inspection and testing center of ministry

of agriculture (Beijing, China). The internal standards, RNZ-

d3 and DMZ-d3, were acquired from RIVM (Bilthoven, The

Netherlands), HMMNI-d3 from WITEGA (Berlin, Germany).

Oasis MCX (60 mg) extraction columns were from Waters

(Milford, MA).

2.2. Equipment

The liquid chromatography mass spectrometer system con-

sisted of a Waters Alliance 2690 LC system equipped with a

Micromass (Manchester, UK) Quattro LC tandem mass spec-

trometer. Positive ions were acquired in the multiple reaction

monitoring (MRM) mode using a desolvation temperature of

300 C and a source temperature of 80 C. Nitrogen was used

as nebulisation and desolvation gas, at flow-rates of 30 and

440Lh1, respectively. For each analyte, the two most abun-

dant product ions were monitored using the conditions given inTable 1. Chromatographic separation was achieved on a Waters

SunFireTM C8 column (100 mm2.1 mm, 3.5m) at a flow-

rate of 0.2 mL min1. The two mobile phases used consisted of

water and acetonitrile. Initial gradient conditions were set to 0%

ACN and held for 0.5 min before incorporating a linear gradient

Table 1

MRM parameters for MS/MS analysis

Compound Transition Dwell

time (s)

Cone

voltage (V)

Collision

energy (eV)

HMMNI 158 > 140 0.1 22 11

158 > 55.1 0.1 22 17

HMMNI-d3 161 0.1 19 13

MNZ 172 > 128 0.1 22 14

172 > 82 0.1 22 23

RNZ 201 > 139.9 0.1 18 11

201 > 55.1 0.1 18 23

RNZ-d3 204 > 143 0.1 20 10

DMZ 142 > 96 0.1 25 15

142 > 81 0.1 25 25

DMZ-d3 145.1 > 99 0.1 25 15

Product ions for quantitation are indicated in bold font.

increasing to 100% ACN at 8 min, and kept at that composition

until 9 min. At 9.1 min the gradient was programmed to initial

conditions to reequilibrate the column for 8.9 min (total run time

18 min). Injection volumes were 10L and all separations were

performed at ambient temperature.

2.3. Sample preparation

About 5 g portions of homogenized liver samples were

added into 50 mL polypropylene centrifuge tubes. Appropriate

amounts of internal standards (2.5g L1 in the final sample

solution) were spiked into each of the samples. About 15 mL of

ethyl acetate were added. After vortexing (2 min) and centrifu-

gation (3000 g, 5 min), supernatant was collected. Repeated

the extraction with 15 mL of ethyl acetate and combined with

the first portion. The organic solutions were evaporated to dry-

ness at 40 C. The residue was dissolved in 5 mL of hexane

and transferred to 10 mL centrifuge tubes. About 3 mL of 0.1 M

HCl was added and vortex-mixed for 10 s. After centrifugation

at 1500 g for 1 min, the hexane layer was discarded and the

aqueous phase was applied to Oasis MCX cartridge, previouslyconditioned with 2 mL of methanol and 2 mL of water. The car-

tridge was then sequentially washed with 2 mL of 0.1 M HCl,

1 mL of methanol and 1 mL of 2% ammonia in MeOHH2O

(10:90 v/v). Analytes were eluted with 2 mL of 2% ammonia in

MeOHH2O (30:70 v/v). The volume of the eluate was reduced

to near 1 mL under nitrogen with a water bath set at 40 C and

reconstituted to 2 mL with water.

2.4. Calibration

Internal matrix standards were used for calibration. Blank

control samples were extracted and cleaned up as described inthe sample preparation section. The matrix-matched blank sam-

ples were spiked with the standards solution in order to obtain

concentrations corresponding to 0, 0.1, 0.5, 1, 5, 10 g kg1 of

tissue for RNZ, MNZ, DMZ, HMMNI and 1g kg1 for three

internal standards.

3. Results and discussion

3.1. Method development

The nitroimidazoles were concentrated and cleaned up by

solid-phase extraction (SPE). The choice of mixed mode SPE

cartridge (MCX) which combines strong cation exchange and

C18 reversed phase interactions was based on previous method

for the determination of nitroimidazoles in porcine urine[23].

Although high recoveries were obtained when the SPE proce-

dure for urine samples was used, significant signal attenuation

resulting from ion suppression was observed comparing with

neat standards. Modifications of the SPE wash solvents and

eluant compositions were tested to isolate the target analytes.

The matrix suppression effect was reduced by the addition of a

wash step with 2% ammonia in 10% methanol and eluting with

2% ammonia in 30% methanol. However, a matrix interference

peak occurred in the control liver sample at the retention time




396 X. Xia et al. / Analytica Chimica Acta 586 (2007) 394398

as MNZ, which was equivalent to



X. Xia et al. / Analytica Chimica Acta 586 (2007) 394398 397

Table 2

Recovery, repeatability and in-house reproducibility in spiked samples

Compound Spike level (g kg1) Recovery (%) Repeatability (%) Reproducibility (%)

HMMNI 0.5/1.0 96.3/95.3 9.9/11.1 11.2/13.5

HMMNI-d3 (ISa)

MNZ 0.1/0.5/1.0 85.1/85.5/83.6 7.8/10.7/4.0 8.2/13.9/7.8

RNZ-d3 (IS)

RNZ 0.1/0.5/1.0 98.3/89.2/84.7 14.1/7.6/7.0 19.2/10.1/8.7

RNZ-d3 (IS)

DMZ 0.1/0.5/1.0 98.8/88.8/87.8 8.2/6.3/7.7 15.4/11.6/9.5

DMZ-d3(IS)

a IS: internal standard.

Table 3

Confirmatory analysis of spiked samples in three recovery studies

Fragment ions Spiked sample Matrix standard

tR(min) Ion ratio tR(min) Ion ratio

First recovery study

HMMNI 55/140 7.16 0.164 0.013 7.15 0.156 0.022

MNZ 82/128 7.35 0.268 0.016 7.35 0.290 0.004

RNZ 55/140 7.54 0.165

0.016 7.52 0.189

0.021DMZ 81/96 8.00 0.192 0.005 8.00 0.191 0.016

Second recovery study

HMMNI 55/140 7.15 0.164 0.026 7.15 0.158 0.023

MNZ 82/128 7.35 0.273 0.019 7.35 0.285 0.021

RNZ 55/140 7.53 0.162 0.030 7.52 0.186 0.033

DMZ 81/96 7.9 0.200 0.005 8.00 0.192 0.011

Third recovery study

HMMNI 55/140 7.16 0.167 0.009 7.16 0.167 0.031

MNZ 82/128 7.35 0.327 0.019 7.35 0.296 0.017

RNZ 55/140 7.52 0.158 0.020 7.54 0.188 0.015

DMZ 81/96 8.01 0.192 0.013 8.00 0.195 0.010

HMMNI spiked at 0.5g kg1, MNZ, RNZ, DMZ spiked at 0.1g kg1.

Table 2, mean recoveries of nitroimidazoles ranged from 83% to

98%. The corresponding variation intra- and inter-day repeata-

bility, expressed as relative standard deviation (R.S.D.), was less

than 14.1% and 19.2%, respectively.

Table 3 shows the confirmatory analysis of spiked samples in

three recovery studies. The retention time (tR) of each analyte

in matrix standards and spiked samples are essentially identi-

cal within instrumental variation. The two transitions for each

compound were used for computing ion ratios, which were then

compared to those produced by matrix standards for confirma-

tion. Theconfirmationratios of spikedsamplesare within10% of

the confirmation ratios of the matrix standards for MNZ, DMZ,

HMMNI and within 20% for RNZ. Intra-day precision of confir-mation ratios was highly reproducible (R.S.D. of



398 X. Xia et al. / Analytica Chimica Acta 586 (2007) 394398

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