Development and validation of a new method for determining nitrofuran metabolites in bovine urine using liquid chromatography - tandem mass spectrometry (LC-MS/MS).

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*Corresponding author: Lech Rodziewicz, Laboratory of Chemical Examination in Foodstuffs, Department of Hygiene Veterinary,

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M&",0S!T'UV$The use of nitrofurans as veterinary drugs in food-producing animals is banned throughout the European

Union. Nevertheless, nitrofuran metabolites have been detected not only in animal products, but also in bovine urine. At present there are no methods yet published for the simultaneous detection of nitrofuran metabolites in bovine urine.

CWXY")/ZY(V$To develop and validate a method for determination of four key nitrofuran metabolites in bovine urine. D&)YS/&-$&'U$[Y)\!U(V$The four nitrofuran metabolites (nitrofurantoin, furazolidone, nitrofurazone and furaltadone), were

determined in bovine urine using LC-ESI-MS/MS. The procedure required an acid-catalysed release of protein-bound metabo-lites, followed by their in situ conversion into 2-nitrobenzaldehyde (NBA) derivatives. The sample clean-up was performed using a polymer extraction cartridge before hydrolysis. Nitrofuran metabolites were then determined using electrospray ionization in the positive mode, that had previously been separated on a Phenomenex Luna C-18 column.

Y(T-)(V$The method was validated in accordance with the procedure outlined in the Commission Decision No. 2002/657/

EC. Urine samples were spiked with nitrofuran metabolite solutions at levels of 0.5, 1.0, 1.5 and 2.0 µg/kg. Recoveries +-,P%$)F%*3%%,);D)Q)8DCR)S",*%+)&*-,$-+$:0!++%0*%$T1)3"*')-)+%(%-*-F"@"*.)(+%0"&"!,)SU5NT)!G)@%&&)*'-,)8;R)G!+)-@@)G!M+) analytes. The decision limit (CC) and detection capability (DC) were obtained from a calibration curve and lay respectively 3"*'",)*'%)G!@@!3",P)+-,P%&V)DW88)Q)DWXB)YPZ>P)-,$)DW8X)Q)DWBX)YPZ>PW

P!'"-T(/!'(V$The developed and validated LC-ESI-MS/MS method allows four nitrofuran metabolites to be identified and

quantitated in bovine urine. This analytical procedure meets the criteria defined in the Commission Decision No. 2002/657/EC.

Key words: 2&3).!$45).!,$.)65&745/.$8!6/2&9)6!:,$5!:)'4!:,$ ;<=>-<?>@?> NF @N+P+@EH@ J]S!*&U#Y'/YV$[,"-)JM+!(%\&>-)]-F+!,"=-)&*!&!3-,"-),"*+!GM+-,^3)\->!)@%>^3)3%*%+.,-+.\,.0')M)]3"%+]_*1)>*^+.0')*>-,>") (+]%],-0]!,%)&_)$!)&(!`.0"-W)a%*-F!@"*.),"*+!GM+-,^3)&_)3.>+.3-,%)]-+^3,!)3)(+!$M>*-0')(!0'!$]%,"-)]3"%+]40%P!)\->)") I!0]MW)<+->)!(MF@">!3-,%\)I%*!$.)(!]3-@-\_0%\),-)\%$,!0]%&,%)!],-0]-,"%)I%*-F!@"*^3),"*+!GM+-,^3)3)I!0]M)F.$@40.IW PY-V$$$H%@%I)F-$-b)F.=!)!(+-0!3-,"%)")]3-@"$!3-,"%)I%*!$.)-,-@"*.0],%\)(!]3-@-\_0%\),-)!],-0]-,"%)0]*%+%0')I%*-F!@"*^3) ,"*+!GM+-,^3)3)I!0]M)F.$@40.IW D&)YS/&^$/$[Y)!U&V$a%*-*-F!@"*.),"*+!GM+-,^3)S,"*+-GM+-,*!",.1)GM+-]!@"$!,M1),"*+!GM+-]!,M)")GM+-@*-$!,MT)F.=.)!],-0]-,%) 3)I!0]M)F.$@40.I)I%*!$_)EH:J5/:a5Za5W)?+!0%$M+-)3.I-P-)&*!&!3-,"-)'.$+!@"].)3)c+!$!3"&>M)>3-c,.I)3)0%@M) +!]%+3-,"-) 3"_]-,"-) F"-=>!:I%*-F!@"*1) -) ,-&*4(,"%) (+]%(+!3-$]%,"-) I%*-F!@"*^3) 3) (!0'!$,%) ]) 6:,"*+!F%,]-@$%'.$%IW) d0].&]0]-,"%)(+^F%>)(+!3-$]!,!)(+]%$)'.$+!@"]_),-)>!@MI"%,>-0')]%)]=!`%I)(!@"I%+.0],.IW)a%*-F!@"*.),"*+!GM+-,^3) !],-0]-,%)F.=.)(+].)]-&*!&!3-,"M)\!,"]-0\")(!(+]%])%@%>*+!+!](+-&]-,"%)3)(!@-+.]-0\")$!$-*,"%\1)(!)30]%c,"%\&].I)+!]-dzielaniu na kolumnie Phenomenex Luna C-18.

J_'/,/V$a%*!$-)]!&*-=-)]3-@"$!3-,-)]P!$,"%)])$%0.]\_)e!I"&\"),+)6DD6Z79fZgJW)?+^F>")I!0]M)F.=.)3]F!P-0!,%)+!]-*3!+%I)I%*-F!@"*^3),"*+!GM+-,^3),-)(!]"!I-0')!$(!3"-$-\_0.0')D191)81D1)8191)")61D)hPZ>PW)d$].&>)I"%c0"=)&"4)3)]->+%&"%) ;D) Q) 8DCR1) 3&(^=0].,,">) (!3*-+]-@,!c0") SU5NT) F.=) I,"%\&].) ,"`) 8;R) $@-) 3&].&*>"0') 0]*%+%0') ]3"_]>^3W) N%0.].\,_) 3-+*!ci)P+-,"0],_)SHH T)!+-])]$!@,!ci)3.>+.0"-)SHH!T)3.],-0]!,!)(+].)M`.0"M)>+].3%\)3]!+0!3%\W)a"%&]0]_)&"4)!,%) w zakresie 0,11 – 0,34 "g/kg oraz 0,13 – 0,43 "g/kg.

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Nitrofurans (NFs) are widely used as antibacterial agents in feed additives for treating gastrointestinal in-fections such as bacterial enteritis caused by =:"#!5)"#)/$ "&9) and >/98&.!99/ in cattle, pigs, farm reared poultry, fish and shrimps. The most common NFs are nitrofuran-toin, furazolidone, nitrofurazone and furaltadone. After administration to animals, these nitrofurans are rapidly metabolized ).$3)3& to more stable products. Thus, the analysis of NFs in biological samples is based on the determination of their metabolites. The marker residues for the nitrofurantoin, furazolidone, nitrofurazone and furaltadone are correspondingly; 1-aminohydantoin SjONT1) X:-I",!:6:!K-]!@"$",!,%) Sjd2T1) &%I"0-+F--zide (SEM) and 3-amino-5-morpholinomethyl-2-oxa-]!@"$",!,%)Sjad2T)k86lW

The EU has however banned NFs from being used in veterinary drugs in animals intended for human consumption because of their proven carcinogenic and genotoxic effects. According to Commission Decision No. 2003/181/EC [3], the minimum required perfor-I-,0%)@%#%@)SaU?ET)'-&)F%%,)&%*)-*)8)YPZ>P)G!+)%-0') of these NF metabolites in poultry meat and aquaculture products, but not up till now in urine. In Poland, NF metabolites are monitored by the authorities as part of the official control on veterinary drug residues.

NFs and their metabolites are excreted mainly by the -,"I-@)@"#%+)-,$)>"$,%.&W)/,$%%$1)jd2)0!,0%,*+-*"!,&) highly correlated (r>0.97) between the liver and kidneys of pigs after the administration of furazolidone at 400 IPZ>P)",)G%%$)G!+)f)$-.&W)m'%)M+",-+.)jd2)'-@G:@"G%)3-&) 14.7 days [7]. These results thereby demonstrated that M+",-+.)jd2)0-,)(+!#"$%)*'%)F-&"&)G!+)I!,"*!+",P)*'%) illegal use of furazolidone in pigs without slaughter, i.e.

by non-invasive means.

To date, several LC-ESI-MS/MS methods have been reported for determining the four NF metabolites in various biological matrices (e.g. muscles, eggs, honey, milk and plasma) [2, 8, 9, 10, 11], and all involve si-multaneous acid hydrolysis and derivatisation of the NF residues with 2-nitrobenzaldehyde (NBA). The diffe-rences between the methods are in the sample clean-up. So far however, there are no LC-ESI-MS/MS methods for determining all four metabolites in animals’ urine. Currently, only single, evaluated analytical methods are available allowing measurement of single NF metabo-lites in animal urine. Two of them, high performance liquid chromatography (HPLC) with UV detection [1] and an immuno-chromatographic method have esti-mated AHD [13], with a limit of detection (LOD) >10 µg/l. The sensitivity of these methods was nevertheless insufficient to reach the MRPL of 1 µg/kg. A third me-thod using enzyme-linked immunoassay (ELISA) was

-@&!)$%#%@!(%$)G!+)*'%)$%*%+I",-*"!,)!G)jd2)",)M+",%) which had an LOD of approximately 1.0 µg/l.

The aim of this study was to therefore develop and validate a method for simultaneously determining the aforementioned metabolites, i.%W) Q)jON1)jd21) 5Ja) -,$)jad2) ",) F!#",%) M+",%) M&",P) -) EH:J5/:a5Za5) technique. The method involved a preliminary clean--up by solid phase extraction (SPE), followed by acid hydrolysis/derivatisation and liquid-liquid extraction. High sensitivity was achieved with tandem mass spec-trometry using API 5500 equipment (AB SCIEX Q TRAP 5500, Canada), providing analyte identification at low concentrations. The method may be used for the detection of all four residues down to 0.5 µg/kg.

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m'%)jON1)jd21) 5Ja) -,$)jad2) I%*-F!@"*%&) were obtained from Witega (Berlin, Germany) and from Sigma-Aldrich (St. Louis, MO, USA). Internal &*-,$-+$&)jd2:$B1)jad2:$9)-,$)6:,"*+!F%,]-@$%'.-de (2-NBA) and the 2-NBA &*-,$-+$&)jd2:$B1)jad2:$9)-,$)6:,"*+!F%,]-@$%'.-derivatives of nitrofuran I%*-F!@"*%&)n?jON1)n?jd21)n?5Ja)-,$)n?jad2) were supplied by Sigma-Aldrich. Strata X (500 mg/3 ml) cartridges were purchased from Phenomenex (Tor-rance, USA). Methanol LC-MS grade, methanol LC grade, ethyl acetate LC grade and hydrochloric acid were provided by Baker (Deventer, The Netherlands). Trisodium phosphate dodecahydrate p.a., sodium hy-droxide p.a. and ammonium acetate p.a. were received from Merck (Darmstadt, Germany). Ultrapure water was supplied by Milli-Q system Millipore (Bedford, MA, USA). The filters for the extract filtration were from Millipore (Millex GV, 0.45 Ym).

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Stock standard solutions of 1.0 mg/ml were prepa-red in calibration flasks by initially dissolving 50 mg !G)jd21)5Ja1)jON)-,$)jad2)",)9D)I@)!G)I%*'-,!@W) These solutions were then diluted 100 times in methanol giving an intermediate standard solution at 10 µg/ml. A working standard solution of 10 ng/ml was then made by appropriate dilution of intermediate standard with I%*'-,!@W)/,*%+,-@)&*-,$-+$&)!G)jd2:$B)-,$)jad2:$9) were prepared by dissolving 10 mg of each in methanol, which were accordingly diluted until a working solution of 10 ng/ml was achieved. The stock standards solutions (1.0 mg/ml) kept at -20ºC were stable for 1 year. The intermediate standard solutions (10 µg/ml) stored at -20ºC were stable for one month. The working standard solutions (10 ng/ml) stored at temperatures in the range of 2 – 8ºC were stable for 1 week.

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m'%)&*!0>)&!@M*"!,&)!G)n?jd21)n?5Ja1)n?jON) -,$)n?jad2)3%+%)(+%(-+%$)",)I%*'-,!@)-*)0!,0%,-trations of 20 µg/ml. Standard solutions were prepared from these stock solutions in acetonitrile : water (1:2; v/v) to give concentrations of 0.5, 1, 1.5 and 2 µg/kg. >/8D9!$D5!D/5/6)&.$

Urine samples were first centrifuged at 3000 rpm for 10 min, then filtered with a 0.45 µm PVDF membrane and stored at -20°C until analysis.

A 1 ml urine sample was then weighed and an ",*%+,-@)&*-,$-+$)I"K*M+%)3-&)-$$%$)S8D),PZI@)!G)jd2-:$B) -,$)jad2:$91) 8DD) h@TW)m'%) &-I(@%) 3-&) (-&&%$) through a Strata X cartridge (500 mg/3 ml), which had been previously pre-conditioned with 3 ml methanol, followed by 3 ml water prior to use. The cartridge was then rinsed with 3 ml water and the rinsed eluates containing the analytes were collected in suitable glass vials. Subsequently, 9 ml hydrochloric acid (0.1 mol/l) and 100 µl 2-NBA (50 mmol/l) were added to the test eluates followed by vortex mixing.

Vials were then capped tightly and thermostatically incubated overnight at 37 ±1ºC after which samples were then cooled to room temperature and neutralised by adding 1.0 ml trisodium phosphate (0.3 mol/l) with the pH adjusted at 7.00 ±0.5 using sodium hydroxide (2 mol/l) as appropriate. From this mixture, 7 ml of supernatant were transferred into suitable glass tubes, placed into a heating block maintained at 40 ±5°C and evaporated to dryness under a stream of nitrogen. The residues were then resuspended in 500 µl of methanol--water (10:90, v/v) followed by vortex mixing for 30 s. The resulting aliquots were transferred into micro centrifuge tubes and spun at 14000 rpm for 5 min followed by filtration through a 0.45 µm nylon filter directly into the LC vial.

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The LC-ESI-MS/MS systemconsisted of an Agilent 1200 HPLC (Agilent Technologies, Germany) connec-ted to a Applied Biosystems/AB SCIEX Q TRAP 5500 linear ion trap mass spectrometer (Concord, Ontario, Canada), coupled to a TurboIonSpray (TISP).

LC analyses were performed on a Luna C18(2) co-lumn (150 × 2 mm, I.D 3 Ym particle size; Phenomenex, Torrance, USA). The column was thermostatted at 40ºC, the flow rate was 200 µl/min, the injection volume was 10 µl and the following programme of mobile phases were M&%$)0!,&"&*",P)!GV)DW9)II!@Z@)-II!,"MI)-0%*-*%)",)6DR) methanol in water (A) and methanol (B). To achieve the required separation, the linear gradient run comprised; DWD)Q)DW8)I",)8DR)j1)DW8)Q)89)I",)G+!I)8D)*!);DR)j1) 89)Q)87)I",)G+!I);DR)*!)8DR)j)-,$)87)Q)69)I",)8DR)jW) The MS was operated in Multiple Reaction Moni-toring mode (MRM). Two MRM transitions were

mo-nitored for each compound. The most intense was used for quantification and the minor one for confirming the analyte identity. Both quantification and identification procedures were optimised. MS tuning was performed in positive ESI mode by infusing solutions of analytes (100 µg/l in methanol) at a flow rate of 10 µl/min mixed with an LC flow comprising solvents A and B (v/v; 0.2 ml/min) using a T-connector. MRM transitions and optimal values for the declustering potential (DP) collision energy (CE) and collision cell exit potential (CXP) are shown in Table 1.

Table 1. Mass spectrometry detection conditions using MRM mode Analytes MRM transition (m/z) Retention time (min) DP (V) CE (V) CXP (V) NPAHD 6B;)o)8XB 6B;)o)8fC 10.1 46 48 21 21 8 8 n?jd2 6X7)o)8XB 6X7)o)8DB 10.5 46 46 14 26 10 10 NPSEM 6D;)o)8;6 6D;)o)877 10.8 36 36 13 10 8 8 n?jad2 XX9)o)6;8 XX9)o)676 13.0 42 42 21 19 10 10 n?jd2:$B)S/5T n?jad2:$9)S/5T 6BD)o)8XB XBD)o)6;7 10.4 12.9 48 46 20 24 12 12 Other ESI parameters used were as follows; ion spray voltage (IS) at 4500 V: source temperature (TEM) at 400ºC: curtain gas (CUR) at 30 psi: CAD gas was medium: ion source gas at 1 (GS1), 40 psi: and gas 2 (GS2) at 70 psi. Nitrogen served both as turbo gas and collision gas. The dwell time was set to 100 ms. Data collection and subsequent processing were performed with Analyst 1.5.2 Software.

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In accordance with the Commission Decision 20002/657/EC, a sample can only be determined posi-tive when the following criteria are met; the signal-to--noise ratio (S/N) of diagnostic ions have to be greater than three, the relative ratio of analyte retention time to that of the IS corresponds to that of the calibration &!@M*"!,)3"*'",)-)p6W9R)*!@%+-,0%)G!+)@"qM"$)0'+!I-*!-graphy as well as for the relative peak area ratios of the various transition reactions within the tolerances set by the EU criteria [4].

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A common approach to analysing NF metabolites in biological samples involves an acid hydrolysis and derivatisation with 2-nitrobenzaldehyde (NBA), whe-re the latter is necessary for the determination of NF

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metabolites. All NF metabolites have low molecular I-&&%&1) G+!I) f9) IZ]) S5JaT) *!) 6D8) IZ]) Sjad2TW) The LC-ESI-MS/MS analysis of low molecular mass substances displays relatively poor sensitivity due to the high abundance of mass spectrometric background noise within this mass range, as well as non-specific fragmentation behaviour. The derivatisation of the NF metabolites with NBA however usually significantly increases the sensitivity of the MS analysis, as has been described in the literature. In the presented paper, derivatisation was performed according to the pro-cedure published by !)6.!5$ et al. [6], although with some modifications. Unfortunately, derivatisation may increase matrix side-reactions which can interfere with subsequent LC-ESI-MS/MS analysis. Removing any drugs from the matrix has been found to be one of the most difficult steps in the analysis of NF metabolites in urine samples. The current study describes such a method for determining NF metabolites in urine where solid-phase extraction (SPE) was used as a clean-up step prior to hydrolysis and derivatisation. The three following polymeric columns for SPE were tested: Strata X (Phenomenex, 500 mg/3 ml), Strata SDB-L (Phenomenex, 200 mg/3ml) and Oasis HLB (Waters Co, 30 mg/3 ml). The clean-up was assessed through recoveries, that were calculated by comparing peak areas of spiked samples (at 1 µg/kg) with corresponding derivatised standards. In these instances, no internal standard was used and quantification was carried out using the external absolute response. The samples were prepared as described in the ‘Sample preparation’ sec-tion. Results so obtained, showed that all three sorbents provided similar recoveries, but that the Strata X gave the greatest reductions of interfering compounds and hence allowed NF metabolites to be determined at low levels. Therefore, the Strata X was selected as the SPE cartridge for the main study. The absolute recoveries G!+) *'%) 5*+-*-) r) 0-+*+"$P%) 3%+%) 77R1) XfR1) 8CR) -,$) 69R)+%&(%0*"#%@.)G!+)n?jON1)n?jd21)n?5Ja1)-,$) n?jad2W)m'%&%)@!3)+%0!#%+"%&)3%+%)$M%)*!)%K*%,&"#%) matrix effects; a common problem of the ESI technique. However, improved and satisfactory recoveries were in fact obtained by quantification using calibration cu-rves based on matrix matched standards and also due to the use of internal standards which compensate for the signal loss resulting from matrix effects as well as the analyte loss during sample preparation; the internal standard being added before sample preparation. In the present study, the relative recoveries were thereby '"P'%+)*'-,);DR)-,$)+%@-*"#%)&*-,$-+$)$%#"-*"!,&)3%+%) F%@!3)8;RW

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The developed method was validated on the basis of procedures described in Commission Decision No.

2002/657/EC [4]. The following performance characte-ristics were checked: specificity, linearity, authenticity (i.e. through recovery), precision (repeatability and wi-thin-laboratory reproducibility), decision limit (CC) and detection capability (DC). The method was validated G!+)*'%)&"IM@*-,%!M&)-,-@.&"&)!G)jON1)jd21)5Ja)-,$) jad2)F.)*'%"+)$%+"#-*"&%$)nL)0!M,*%+(-+*&s)n?jON1) n?jd21)n?5Ja)-,$)n?jadjW

The specificity of the method was demonstrated by analysing 20 different blank and spiked urine sam-ples at 0.5 µg/kg in order to investigate any possible interferences that had similar retention times with the analytes. For each of the transitions, chromatograms demonstrated a significant increase in peak area and intensity at their specific retention time compared to the blanks, taking into account a signal to noise ratio of at least 3. Figure 1 shows the LC-ESI-MS/MS chroma-tograms of blank and spiked urine; at a concentration of 0.5 µg/kg. In the blank samples, interfering peaks were observed at the SEM retention time for the 209192 and 209166 ion transitions. However, when comparing chromatograms of blank bovine urine with the bovine urine spiked with 0.5 µg/kg, the SEM clearly shows that the interference is minimal and of little consequence since the interference signal is very low compared to that obtained from the positive sample, even at its low levels. All the chromatograms obtained throughout the validation study demonstrated very stable retention times for the spiked samples, with relative deviation -@3-.&)F%",P)F%**%+)*'-,)p619RW

A minimum of four identification points are required for the identification of banned substances (according to Decision No. 2002/657/EC). This is fulfilled using a LC-ESI-MS/MS method with 1 precursor ion (1 IP) and at least 2 transition product ions (2×1.5 IP). Pre-cursor (1 IP) and product ions (1.5 IP) of each analyte are presented in Table 1.

The linearity of the developed method was eva-luated for each of the NF metabolites by preparing calibration curve blank matrices, which consisted of six

Table 2. Recovery, repeatability and reproducibility in spi-ked bovine urine samples (n=6)

Analytes Parameter Level of spiking ("g/kg) 0.5 1.0 1.5 2.0 AHD Recovery ("g/kg) U%(%-*-F"@"*.)SU5N1RT U%(+!$M0"F"@"*.)SU5N1RT 101.7 10.8 13.9 99.2 8.6 10.1 103.4 7.5 10.7 102.5 7.8 8.2 jd2 Recovery ("g/kg) U%(%-*-F"@"*.)SU5N1RT U%(+!$M0"F"@"*.)SU5N1RT 102.3 9.7 11.3 102 7.6 9.3 92.8 6.8 8.1 94.1 6.1 7.3 SEM Recovery ("g/kg) U%(%-*-F"@"*.)SU5N1RT U%(+!$M0"F"@"*.)SU5N1RT 101.9 18.8 23.6 99.4 15.8 21.1 102.5 15.8 20.9 107.5 14.5 18.7 jad2 Recovery ("g/kg) U%(%-*-F"@"*.)SU5N1RT Reproducibility (RSD) 90.4 8.9 9.7 94.1 6.5 9.4 97.5 6.3 8.7 103.2 5.3 6.1

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different bovine urine matrices. The range of the cali-bration curves were from 0.0 (blank sample) to 2 µg/kg (i.e. at the following points; 0.0, 0.5, 1.0, 1.5 and 2.0 µg/ >PTW)j)G"K%$)-I!M,*)!G)",*%+,-@)&*-,$-+$)Sjd2:$B)-,$) jad2:$9T)3-&)-$$%$)*!)-@@)*'%)&-I(@%&W)m'%)jd2:$B) 3-&)M&%$)G!+)jON1)jd2)-,$)5Ja)3'"@&*)jad2:$9) G!+)jad2W)H-@"F+-*"!,)0M+#%&)3%+%)0!,&*+M0*%$)M&",P) analyte/internal standard peak area ratios versus analyte concentration. Adequate linearity was obtained for each analyte with a correlation coefficient higher than 0.993 and a slightly inferior but still acceptable SEM of 0.985.

The measurement accuracy (i.e. corrected recove-ry of internal standard), was assessed by spiking six

replicates of blank urines for each standard level; 0.5, 1.0, 1.5 and 2.0 µg/kg. The results are listed in Table 2. The average recoveries for each compound ranged G+!I);DR)*!)8DCR)-,$)GM@G"@)*'%)0+"*%+"-)(M*)G!+3-+$)",) the 2002/657/EC Decision stating that a mass fraction, which is less than or equal to 1 µg/l should give a mean +%0!#%+.)!G)9D)Q)86DR1)3'"@%)fD)Q)88DR)!G)+%0!#%+.) should be obtained when the mass fraction ranges from 1 to 10 µg/kg. The recoveries obtained by this analytical method were therefore highly satisfactory.

The precision (repeatability and within-laboratory reproducibility) of the assay was determined by the relative standard deviation (RSD). The repeatability of

XIC of +MRM (11 pairs): 209.000/192.000 Da ID: SEM from Sample 3 (Sample003) of Mocz12.11.12.wiff (Turbo Spray) Max. 3240.0 cps.

9.0 9.5 10.0 10.5 11.0 11.5 12.0 12.5 13.0 13.5 14.0 Time, min 0 200 400 600 800 1000 1200 1400 1600 1800 2000 2200 2400 2600 2800 3000 3200 In te ns ity , c ps 12.61 10.24 13.33 11.97 13.84 13.97 10.81 11.17 11.39 9.40 11.08 !"#$%$&'& SEM 209-192

9.0 9.2 9.4 9.6 9.8 10.010.210.410.610.811.011.211.411.611.812.012.212.412.612.813.013.213.413.613.814.0 Time, min 0 200 400 600 800 1000 1200 1400 1600 1800 2000 2200 2400 2600 2800 3000 3200 3400 3600 3800 4000 4200 4340 In te ns ity , c ps 10.81 12.59 10.28 13.19 11.94 11.45 9.90 11.16 9.18 9.40 !"#$%$&(') SEM 209-192

9.0 9.2 9.4 9.6 9.8 10.010.210.410.610.811.011.211.411.611.812.012.212.412.612.813.013.213.413.613.814.0 Time, min 0 100 200 300 400 500 600 700 800 900 1000 1100 1200 1300 1400 1500 1600 1700 1800 1900 2000 2100 2200 2300 In te ns ity , c ps 11.11 10.81 9.89 9.83 11.41 11.47 9.61 13.98 11.7111.81 13.68 9.56 12.13 13.32 9.23 12.7212.9113.17 9.11 12.64 !"#$%$/'& SEM 209-166

9.2 9.4 9.6 9.810.010.210.410.610.811.011.211.411.611.812.012.212.412.612.813.013.213.413.613.814.014.2 Time, min 0 200 400 600 800 1000 1200 1400 1600 1800 2000 2200 2400 2600 2800 3000 3200 3400 3580 In te ns ity , c ps 10.81 10.26 11.13 9.86 11.32 9.70 11.41 12.36 13.72 11.91 9.48 11.69 11.97 12.70 12.76 13.3213.36 9.38 9.08 13.97 !"#$%$0'1 SEM 209-166

XIC of +MRM (11 pairs): 236.000/134.000 Da ID: AOZ from Sample 3 (Sample003) of Mocz12.11.12.wiff (Turbo Spray) Max. 9750.0 cps.

9.2 9.4 9.6 9.810.010.210.410.610.811.011.211.411.611.812.012.212.412.612.813.013.213.413.613.814.0 Time, min 0 500 1000 1500 2000 2500 3000 3500 4000 4500 5000 5500 6000 6500 7000 7500 8000 8500 9000 9500 9750 In te ns ity , c ps 12.97 13.88 10.48 9.51 12.66 AOZ 236-134

XIC of +MRM (11 pairs): 236.000/134.000 Da ID: AOZ from Sample 4 (Sample004) of Mocz12.11.12.wiff (Turbo Spray) Max. 2.1e4 cps.

9.2 9.4 9.6 9.8 10.010.210.410.610.811.011.211.411.611.812.012.212.412.612.813.013.213.413.613.814.0 Time, min 0.0 1000.0 2000.0 3000.0 4000.0 5000.0 6000.0 7000.0 8000.0 9000.0 1.0e4 1.1e4 1.2e4 1.3e4 1.4e4 1.5e4 1.6e4 1.7e4 1.8e4 1.9e4 2.0e4 2.1e4 In te ns ity , c ps 10.48 13.01 13.82 !"#$%$&2 AOZ 236-134

9.0 9.2 9.4 9.6 9.810.010.210.410.610.811.011.211.411.611.812.012.212.412.612.813.013.213.413.613.814.0 Time, min 0.0 1000.0 2000.0 3000.0 4000.0 5000.0 6000.0 7000.0 8000.0 9000.0 1.0e4 1.1e4 1.2e4 1.3e4 1.4e4 1.5e4 1.6e4 In te n si ty , cp s 12.98 11.05 10.48 10.40 9.68 9.96 13.85 AOZ 236-104

9.2 9.4 9.6 9.8 10.010.210.410.610.811.011.211.411.611.812.012.212.412.612.813.013.213.413.613.814.0 Time, min 0.0 2000.0 4000.0 6000.0 8000.0 1.0e4 1.2e4 1.4e4 1.6e4 1.8e4 2.0e4 2.2e4 2.4e4 2.6e4 2.8e4 3.0e4 In te n si ty , cp s 10.48 !"#$%$11'1 AOZ 236-104 XIC of +MRM (11 pairs): 240.000/134.000 Da ID: AOZd4 from Sample 3 (Sample003) of Mocz12.11.12.wiff (Turbo Spray) Max. 6.1e4 cps.

9.0 9.2 9.4 9.6 9.810.010.210.410.610.811.011.211.411.611.812.012.212.412.612.813.013.213.413.613.814.0 Time, min 0.0 5000.0 1.0e4 1.5e4 2.0e4 2.5e4 3.0e4 3.5e4 4.0e4 4.5e4 5.0e4 5.5e4 6.0e4 In te n si ty , cp s 10.36 !"#$%$34') AOZ-d4 240-134

XIC of +MRM (11 pairs): 240.000/134.000 Da ID: AOZd4 from Sample 4 (Sample004) of Mocz12.11.12.wiff (Turbo Spray) Max. 6.8e4 cps.

9.2 9.4 9.6 9.8 10.010.210.410.610.811.011.211.411.611.812.012.212.412.612.813.013.213.413.613.814.0 Time, min 0.0 5000.0 1.0e4 1.5e4 2.0e4 2.5e4 3.0e4 3.5e4 4.0e4 4.5e4 5.0e4 5.5e4 6.0e4 6.5e4 6.8e4 In te n si ty , cp s 10.38 !"#$%$444'4 AOZ-d4 240-134 a b !"#$%&'&()&!&*+,&& -./012&31431&56$78)97"$)8:&7;&*<)=>&()+&)=?&:@!>%?&(*+&#$!=%&:)8@<%:&)9&A,B&C"4>"

(6)

the method was evaluated by spiking blank samples of urine in three sets, each consisting of six replicates at levels of 0.5, 1.0, 1.5 and 2.0 µg/kg. These were ana-lysed on different days by the same instrument and the same operator. The within-laboratory reproducibility was calculated identically except that analyses were performed by different operators. The results are shown in Table 2. Satisfactory repeatability and within-labora-97$D&$%@$7?#5!*!<!9D&E%$%&7*9)!=%?&;7$&FGHI&FJK&)=?& F3JK&)9&)<<&<%L%<:M&N1H:&E%$%&<7L%$&96)=&'OP&)=?& QBPI&$%:@%59!L%<D,&

7$&103I&$%@%)9)*!<!9D&)=?&E!96!=-XIC of +MRM (11 pairs): 249.000/134.000 Da ID: AHD from Sample 3 (Sample003) of Mocz12.11.12.wiff (Turbo Spray) Max. 1.0e4 cps.

9.2 9.4 9.6 9.810.010.210.410.610.811.011.211.411.611.812.012.212.412.612.813.013.213.413.613.814.0 Time, min 0.00 500.00 1000.00 1500.00 2000.00 2500.00 3000.00 3500.00 4000.00 4500.00 5000.00 5500.00 6000.00 6500.00 7000.00 7500.00 8000.00 8500.00 9000.00 9500.00 1.00e4 In te n si ty , cp s 13.27 11.86 12.19 13.02 9.96 10.45 11.30 13.74 9.70 9.40 11.55 AHD 249-134

XIC of +MRM (11 pairs): 249.000/134.000 Da ID: AHD from Sample 4 (Sample004) of Mocz12.11.12.wiff (Turbo Spray) Max. 1.4e4 cps.

9.2 9.4 9.6 9.8 10.010.210.410.610.811.011.211.411.611.812.012.212.412.612.813.013.213.413.613.814.0 Time, min 0.0 1000.0 2000.0 3000.0 4000.0 5000.0 6000.0 7000.0 8000.0 9000.0 1.0e4 1.1e4 1.2e4 1.3e4 1.4e4 In te n si ty , cp s 10.11 13.26 11.87 12.19 13.05 11.23 13.75 9.84 9.58 !"#$#%&'( AHD 249-134

XIC of +MRM (11 pairs): 249.000/178.000 Da ID: AHD from Sample 3 (Sample003) of Mocz12.11.12.wiff (Turbo Spray) Max. 1460.0 cps.

9.2 9.4 9.6 9.810.010.210.410.610.811.011.211.411.611.812.012.212.412.612.813.013.213.413.613.814.0 Time, min 0 100 200 300 400 500 600 700 800 900 1000 1100 1200 1300 1400 1460 In te n si ty , cp s 13.34 12.92 14.12 12.88 9.35 9.60 10.22 11.61 11.92 12.19 13.49 9.21 9.769.8210.16 10.3410.53 11.15 11.41 11.77 12.33 9.08 AHD 249-178

XIC of +MRM (11 pairs): 249.000/178.000 Da ID: AHD from Sample 4 (Sample004) of Mocz12.11.12.wiff (Turbo Spray) Max. 1650.0 cps.

9.2 9.4 9.6 9.8 10.010.210.410.610.811.011.211.411.611.812.012.212.412.612.813.013.213.413.613.814.0 Time, min 0 100 200 300 400 500 600 700 800 900 1000 1100 1200 1300 1400 1500 1600 1650 In te n si ty , cp s 13.35 10.12 12.89 14.02 13.68 9.169.329.429.589.68 10.4410.6410.71 11.2311.2911.4311.5611.7111.8112.2312.36 !"#$#(') AHD 249-178

XIC of +MRM (11 pairs): 335.000/291.000 Da ID: AMOZ from Sample 3 (Sample003) of Mocz12.11.12.wiff (Turbo Spray) Max. 4340.0 cps.

9.2 9.4 9.6 9.8 10.010.210.410.610.811.011.211.411.611.812.012.212.412.612.813.013.213.413.613.8 Time, min 0 50 100 150 200 250 300 350 400 450 500 550 600 650 700 750 800 850 900 950 1000 1050 In te n si ty , cp s 10.18 9.81 10.54 11.34 12.25 10.71 10.85 11.84 13.26 13.78 AMOZ 335-291

XIC of +MRM (11 pairs): 335.000/291.000 Da ID: AMOZ from Sample 4 (Sample004) of Mocz12.11.12.wiff (Turbo Spray) Max. 1.2e4 cps.

9.0 9.2 9.4 9.6 9.8 10.010.210.410.610.811.011.211.411.611.812.012.212.412.612.813.013.213.413.613.814.0 Time, min 0.00 500.00 1000.00 1500.00 2000.00 2500.00 3000.00 3500.00 4000.00 4500.00 5000.00 5500.00 6000.00 6500.00 7000.00 7500.00 8000.00 8500.00 9000.00 9500.00 1.00e4 1.05e4 1.10e4 1.15e4 1.20e4 In te n si ty , cp s 13.00 9.129.30 10.37 10.59 11.33 11.77 12.12 13.31 !"#$#*&'% AMOZ 335-291

9.2 9.4 9.6 9.8 10.010.210.410.610.811.011.211.411.611.812.012.212.412.612.813.013.213.413.613.8 Time, min 0 50 100 150 200 250 300 350 400 450 500 550 600 650 700 750 800 850 900 950 1000 1050 In te n si ty , cp s 13.01 13.30 12.17 9.62 11.86 9.20 10.1010.2010.4910.66 10.93 13.87 AMOZ 335-262

9.0 9.2 9.4 9.6 9.810.010.210.410.610.811.011.211.411.611.812.012.212.412.612.813.013.213.413.613.814.0 Time, min 0 500 1000 1500 2000 2500 3000 3500 4000 4500 5000 5500 6000 6500 7000 7500 8000 8500 9000 9500 In te n si ty , cp s 12.99 !"#$#&% AMOZ 335-262

XIC of +MRM (11 pairs): 340.000/296.000 Da ID: AMOZd5 from Sample 3 (Sample003) of Mocz12.11.12.wiff (Turbo Spray) Max. 2.4e4 cps.

9.2 9.4 9.6 9.8 10.010.210.410.610.811.011.211.411.611.812.012.212.412.612.813.013.213.413.613.8 Time, min 0.0 1000.0 2000.0 3000.0 4000.0 5000.0 6000.0 7000.0 8000.0 9000.0 1.0e4 1.1e4 1.2e4 1.3e4 1.4e4 1.5e4 1.6e4 1.7e4 1.8e4 1.9e4 2.0e4 2.1e4 2.2e4 2.3e4 2.4e4 In te n si ty , cp s 12.88 11.43 !"#$#+)', AMOZ-d5

XIC of +MRM (11 pairs): 340.000/296.000 Da ID: AMOZd5 from Sample 4 (Sample004) of Mocz12.11.12.wiff (Turbo Spray) Max. 7.0e4 cps.

9.0 9.2 9.4 9.6 9.8 10.010.210.410.610.811.011.211.411.611.812.012.212.412.612.813.013.213.413.613.814.0 Time, min 0.0 5000.0 1.0e4 1.5e4 2.0e4 2.5e4 3.0e4 3.5e4 4.0e4 4.5e4 5.0e4 5.5e4 6.0e4 6.5e4 7.0e4 In te n si ty , cp s 12.89 !"#$#,+'-AMOZ-d5 /<)*7$)97$D&$%@$7?#5!*!<!9D&(N1H+&E%$%&*%<7E&'RP&)=?& QOPI&$%:@%59!L%<D,&

The precision (RSD) outcome was not evaluated by the !"#$%& equation because too high values would be obtained due to the concentration range used being too low. However, in accordance with Decision 2002/657/ EC, the RSD obtained for mass fraction lower than 100 µg/kg were as low as possible.

The CC and DC values were determined by the matrix calibration curve procedure according to case 1 of ISO 11843-2 – constant standard deviation [5].

a b

(7)

The CC and DC were calculated using two calibration 5#$L%:&()9&;7#$&<%L%<:&A,AI&A,BI&',AI&',B&)=?&Q,A&C"4>"+I& from six different experiments on different bovine uri-ne matrices performed on different days. Curves were constructed using analyte/internal standard peak area ratio versus analyte concentration. Calculated CC and DC for different NF metabolites are reported in Table S,& T6%& 8%)=& ..U& E%$%& A,''& V& A,SO& C"4>"& )=?& H.W& A,'S&V&A,OS&C"4>",&T6%:%&$%:#<9:&)$%&:)9!:;)597$D&:!=5%& 96%&3NX-&!:&;!Y%?&)9&'&C"4>",

Table 3. CC and CC! obtained in bovine urine

Analytes CC ("g/kg) CC! ("g/kg)

AHD 0.27 0.34

FJK 0.11 0.13

SEM 0.34 0.43

F3JK 0.14 0.18

The analytical method developed was used to de-termine the NF metabolites in 26 bovine urine samples. The majority of the studied compounds were however #=?%9%59)*<%&!=&96%:%&)=)<D:%?&:)8@<%:,&J=<D&FJK&E):& ;7#=?& !=& 7=%& #$!=%& :)8@<%& )9& ',Z& C"4>"& E!96& )& N1H& (=[S+&7;&B,\P,

!" #$%&!"%

The presented LC-ESI-MS/MS method allows si-multaneous determination of four nitrofuran metabolite residues in bovine urine. Solid phase extraction with a Strata X cartridge was used to remove interfering sub-stances from urine samples. This method was validated in accordance with Decision 2002/657/EC and is now used for routine analysis at our laboratory. The repe-atability and laboratory reproducibility of the method E%$%&<%::&96)=&'RP&)=?&QOPI&$%:@%59!L%<D,&T6%&8%)=& values of decision limit and detection capability were *%<7E&'&C"4>",

Conflict of interest

'()*+,%(!"-*.)/0+")*1!*/!120$/%*!2*$1%)")-%3

'()('(" (%

1. 4"+1/$5$+*637*6+0.)""+8+*937*9+.+"$+:+*437*;+"!0$1+* M.: Extraction of nitrofurantoin and its toxic metabolite from urine by supercritical fluids. Quantitation by high performance liquid chromatography with UV detection. Talanta 2003;61:377-383.

2. <!/=*;37*>%+/()0*;37*?!#$=*@.: Validation of a confir-matory method for the determination of resi dues of four

nitrofurans in egg by liquid chromatog raphy-tandem mass spectrometry with the software InterVal. Anal Chim Acta 2007;586:348-358.

3. Commission Decision 2003/181/EC of 13 March 2003 amending Decision No 2002/657/EC as regards the set-ting of minimum required performance limits (MRPLs) for certain residues in food of animal origin. Off J Eur Commun 2003;L71:17-18.

4. Commission Decision 2002/657/EC of 12 August 2002 implementing Council Directive 96/23/EC concerning the performance of analytical methods and interpretation of results. Off J Eur Commun 2002;L221:8-36. 5. ISO 11843-2: Capability to Detection (Part I); Terms

definitions (Part 2), Methodology in the linear calibration case. 2003.

6. A)$%1)"* 437* BC001)"* @37* A$1.1)"* D.: Determination of metabolites of nitrofuran antibiotic in animal tissue by high-performance liquid chromatography – tandem mass spectrometry. J Chromatogr A 2001;939:49-58. 7. A$,*E37* ,+1:*A37*D+1:*E37*E+1:*<.: Tissue depletion and

concentration correlations between edible tissues and biological fluids of 3-amino-2-oxazolidinone in pigs fed with a furazolidone-medicated feed. J Agric Food 2010;58:6774-678 .

8. F+.!G1$=!G$/*437*9!0!1)H*937*<H"1)*@37*I+1+()"*9.: De-tection of banned nitrofuran metabolites in animal plasma using UHPLC-MS/MS. J Chromatogr B 2011;879:159-166.

9. F!.&$)#$/&*A37*B+#+.&=+*J.: Determination of nitrofuran metabolites residues in animal tissues by LC-MS/MS 8%967?,&N75]&X)=:9E&K)><&G!"&QAA\MBZ(O+^_QB/_SQ&(!=& Polish).

10. F!.&$)#$/&* A.: Determination of nitrofuran metabo-lites in milk by liquid chromatograph –electrospray ionization tandem mass spectrometry. J Chromatogr B 2008;864:156-160.

11. '"$5+0+%* A37* @+$--)* K37* I)--+0/)-* ?3: Advantages of LC-MS-MS compared to LC-MS for determination on ntrofuran residues in honey. Anal Bioanal Chem 2006;386:2161-2168.

12. 6)".!1* L37* ;!,).!"* @37* >+1.)"-* @.: Multi-residue monitoring for the simultaneous determination of five nitrofurans (furazolidone, furaltadone, nitrofurazone, nitrofurantoine, nifursol) in poultry muscle tissue thro-#"6&96%&?%9%59!7=&7;&96%!$&;!L%&8)`7$&8%9)*7<!9%:&(FJKI& F3JKI&103I&FGHI&Ha1FG+&*D&<!b#!?&56$7&8)97"$)@6D& coupled to electrospray tandem mass spectrometry – In--house validation in line with Com mission Decision No 657/2002/EC. Anal Chim Acta 2007;586:336-347. 13. E$* @$1:* M37* A$* N$+1:* A37* N$,* >()1* A37* ;($* O+1:* @.:

Development of an immunochromatographic assay for rapid detection of 1-aminohydantion in urine specimens. Biomedical Chromatogr 2009;23:308-314.

Received: 07.03.2013 Accepted: 14.10.2013