Food Chemistry 133 (2012) 536–543
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Analytical Methods
Development and validation of a liquid chromatographic-tandem mass spectrometric method for determination of eleven coccidiostats in milk Szilárd Nász a, Lajos Debreczeni b, Tamás Rikker c, Zsuzsanna Eke a,c,⇑ a
Joint Research and Training Laboratory on Separation Techniques, Eötvös Loránd University, 1117 Budapest, Pázmány Péter Sétány 1/A, Hungary Central Agricultural Office, Food and Feed Safety Directorate, Feed Investigation National Reference Laboratory, 1144 Budapest, Remény utca 42, Hungary c Wessling International Research and Educational Center, 1047 Budapest, Fóti út 56, Hungary b
a r t i c l e
i n f o
Article history: Received 16 March 2011 Received in revised form 12 October 2011 Accepted 14 January 2012 Available online 25 January 2012 Keywords: LC–MS–MS Coccidiostats Milk Validation Solid phase extraction
a b s t r a c t A reversed phase liquid chromatographic–tandem mass spectrometric method with simple solvent extraction and purification by solid phase extraction (SPE) has been developed for the determination of coccidiostats in milk. For sample preparation matrix solid phase dispersion, extraction by organic solvent and SPE with different cartridges were also tested. The compounds determined include lasalocid, narasin, salinomycin, monensin, semduramicin, maduramicin, robenidine, decoquinate, halofuginone, nicarbazin and diclazuril. Main steps of the method are addition of acetonitrile to the milk samples, centrifugation, removal of matrix by SPE, concentration by evaporation and LC–MS–MS determination. During a 15 min time segmented chromatographic run compounds are ionised either positively or negatively. Calculated recoveries range between 77.1% and 118.2%. Maximum levels are in the range of 1–20 lg/kg. The developed method was validated in line with the requirements of Commission Decision 2002/657/EC (2002). It is applicable for control of coccidiostat residues in milk as indicated in Regulation 124/2009/EC (2009). Ó 2012 Elsevier Ltd. All rights reserved.
1. Introduction Coccidiostats are widely used veterinary pharmaceuticals intended to kill or inhibit protozoa. The usage of anticoccidial drugs as feed additives was licensed by the European Union in 2003 (1831/2003/EC, 2003) to allow for the prevention of coccidiosis, a disease that may cause serious economical consequences. Rules of utilisation are specified for different groups of animals. Nevertheless, the so-called unavoidable carry-over of substances into non-target feed may result in undesirable contaminants in derived food of animal origin. Hence maximum levels (ML) were established for 11 coccidiostats in 2009 (124/2009/EC, 2009). Coccidiostats can be classified into two groups. Robenidine, decoquinate, halofuginone, nicarbazin and diclazuril are chemically produced drugs. Lasalocid, narasin, salinomycin, monensin, semduramicin and maduramicin are ionophores. Their chemical structure and polarity varies greatly, particularly in the group of the chemically produced ones. Furthermore nicarbazin is an equimolar complex of 1,3-bis(4-nitrophenyl)urea and 4,6dimethyl-1H-pyrimidin-2-one. These compounds are also known ⇑ Corresponding author at: Joint Research and Training Laboratory on Separation Techniques, Eötvös Loránd University, 1117 Budapest, Pázmány Péter Sétány 1/A, Hungary. Tel.: +36 30 598 0300. E-mail addresses: [email protected] (S. Nász), [email protected] (Z. Eke). 0308-8146/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved. doi:10.1016/j.foodchem.2012.01.022
as 4,40 -dinitrocarbanilide (DNC) and 2-hydroxy-4,6-dimethylpyrimidine (DHP), respectively. DHP excretes fast therefore it is less likely to appear in any food of animal origin. Thus it is reasonable to measure only DNC as a representative of nicarbazin. Several papers describe methods for the determination of coccidiostats. Due to the high boiling point of most coccidiostats liquid chromatography is preferred, usually coupled to tandem mass spectrometry (LC–MS–MS) (Shao et al., 2009; Thompson, Noot, & Kendall, in press; Vincent, Ezerskis, Chedin, & von Holst, 2011; Yakkundi, Cannavan, Elliott, Lövgren, & Kennedy, 2003). UV (Dusi & Gamba, 1999), fluorescence (Matabudul, Crosby, Lumley, & Sumara, 2001) and single quadrupole mass spectrometric detection (Huebra, Vincent, & von Holst, 2010) may also be suitable. Many of these methods are applicable for only a few coccidiostats. Mortier, Daeseleire, and Peteghem (2005a) described a method for five chemical coccididiostats and one metabolite in poultry eggs and feed. Dusi and Gamba (1999) determined four ionophore compounds in poultry feeds, whereas Vincent, Chedin, Yasar, and von Holst (2008) developed a method for six ionophores in poultry and cattle compound feed. Methods for individual compounds like halofuginone (Yakkundi et al., 2003), decoquinate (Feás et al., 2010), diclazuril (Mortier, Daeseleire, & Peteghem, 2005b), nicarbazin (Cannavan, Ball, & Kennedy, 1999; Dusi, Faggionato, Gamba, & Baiguera, 2000), lasalocid (Matabudul et al., 2001), monensin (Chéneau et al., 2007) and semduramicin (Huebra et al., 2010) in different matrices are described as well. In contrast to that only a
S. Nász et al. / Food Chemistry 133 (2012) 536–543
few multi-class methods have been developed for coccidiostats (Cronly et al. 2010; Dubois, Pierret, & Delahaut, 2004; Dubreil-Chéneau, Bessiral, Roudaut, Verdon, & Sanders 2009; Olejnik, Szprengier-Juszkiewicz, & Jedziniak, 2009) and to our knowledge none for milk samples. The maximum levels and maximum residue limits (MRL) for milk given in 124/2009/EC (2009) and 2377/90/EC (1990) are in the range of 1–20 lg/kg except for diclazuril. Since according to 2377/90/EC (1990) this compound is not subject to MRL in ruminants, a reference concentration of 5 lg/kg was used arbitrarily throughout this study. Determination of residues at this concentration level requires the most selective and sensitive detection. Thus triple quadruple detection and multiple reaction monitoring (MRM) are indispensable. For sample preparation matrix solid phase dispersion (MSPD), extraction by organic solvent without any further purification and solid phase extraction (SPE) with different cartridges have also been tested. The developed method is fully validated according to 2002/657/EC (2002) and allows the determination of coccidiostats at trace level. 2. Materials and methods 2.1. Reagents and chemicals Narasin, maduramicin, decoquinate, DNC, DNC-d8, robenidined8, decoquinate-d5 and nigericin were purchased from Sigma (Budapest, Hungary). Lasalocid, salinomycin, monensin, robenidine, halofuginone and diclazuril were purchased from LGC (Wesel, Germany) and semduramicin from Phibro (Ridgefield Park, USA). Diclazuril-bis was kindly donated by Janssen (Beerse, Belgium). Formic acid (98–100%) was from Merck (Budapest, Hungary). For matrix solid phase dispersion Isolute C18 (EC) and for solid phase extraction Oasis HLB (60 mg), Agilent OPT (60 mg), Agilent C-18 (EC) (200 mg), Agilent C-18 (EC) (500 mg), Strata-X (200 mg) were used. All the solvents used were of analytical grade. Water was purified by Milli-Q system (Milli-Q, USA). Analytical grade sodium-formiate was from Reanal (Budapest, Hungary). Stock solutions, containing 1 mg/ml of coccidiostats, were prepared individually. Maduramicin, narasin, nigericin, monensin, salinomycin, and semduramicin were dissolved in methanol, DNC, DNC-d8, diclazuril, diclazuril-bis, robenidine, robenidine-d8 in dimethylsulfoxid (DMSO), decoquinate and decoquinate-d5 in acetonitrile with 6% formic acid. Halofuginone and lasalocid were purchased as 100 lg/ml solutions in acetonitrile. Working solutions were prepared by diluting the individual stock solutions with acetonitrile. Table 1 shows the concentrations of mixed working solution I (target compounds) and II (ISTDs). 2.2. Milk samples For development and validation purposes bovine milk samples with different fat content (1.5%, 2.8%, 3.5%) were obtained from Hungarian retail markets. No residues of target analytes were found in these samples either by the analysis of acetonitrile extract of milk samples or by preparation of milk samples according to the presented procedure thus they were used as blank matrices. 2.3. Instrumentation Agilent 1200 series HPLC consisting of a degasser (G1379B), binary pump (G1312A), injector (G1367B), thermostat (G1330B) and column thermostat compartment (G1316A) coupled to Agilent 6460 triple quadrupole mass spectrometer (G6460A) equipped with Agilent Jet Stream electrospray ion source was used. The instrument was controlled and the results were evaluated by
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MassHunter software. Hermile (Z230A) system was used for centrifugation and a Barkley (Germany) device for evaporation. Visiprep (Supelco) was used for solid phase extraction. 2.4. Chromatographic conditions Separation was achieved using an Agilent Eclipse XDB-C8 (3 150, 3.5 lm) analytical column protected by a Zorbax Eclipse Plus C8 (2.1 12.5 mm, 5 lm) guard column. The oven temperature was set at 30 °C. Eluent A was Milli-Q water, B was acetonitrile, both acidified with 0.1% formic acid. The flow rate was 0.5 ml/min with the following gradient programme: 5% acetonitrile for 1 min, then linear increase of acetonitrile to 95% over 3 min, finally holding on for 11 min. Five minute equilibration time was applied between runs. The injection volume was 30 ll with needle wash in acetonitrile in order to avoid cross-contamination. 2.4.1. Mass spectrometric parameters Jet Stream source parameters were optimised to give the most abundant ion responses for the analytes. Gas temperature was set to 350 °C, gas flow to 10 l/min, nebulizer to 40 psi, sheath gas temperature to 350 °C, sheath gas flow to 7 l/min, capillary voltages, positive and negative alike to 3500 V and nozzle voltages to 1000 V. Multiple reaction monitoring was used for quantification of the compounds because this methodology is the most selective and sensitive detection when applying triple quadrupole mass spectrometry. Individual standard solutions (1 lg/ml in acetonitrile: Milli-Q 50:50) were injected to optimise the transition parameters. As required by the Commission Decision 2002/657/ EC (2002) two transitions were chosen for each coccidiostats. Table 1 shows the chosen transitions as well as the optimised fragmentor voltages and collision energies. The chromatographic run was divided into three time segments: from 0 to 6.3 min positive ion mode, in the second segment from 6.3 to 7.1 min negative ion mode and in the third segment from 7.1 to 15 min positive ion mode was applied. 2.5. Sample preparation 2.5.1. Matrix solid phase dispersion 2 g MSPD sorbent was placed in a mortar and 0.5 ml milk sample spiked at 100 ng/ml was added. It was homogenised with a pestle for 30 s. A syringe barrel containing a filter at the bottom was filled in with the mixture. Six parallels were prepared and the compounds were eluted with different solvents: acetonitrile, methanol, ethyl-acetate, dichloromethane, acetone and hexane. The volume of the solvents was 8 ml in each case. Elutants were collected in glass vials and evaporated to dryness under continuous N2 stream at ambient temperature. The residues were then reconstituted in 500 ll of acetonitrile: Milli-Q water (50:50) and vortex mixed for 30 s prior to injection. 2.5.2. Solvent extraction Two milliliter aliquots of milk samples were placed in centrifuge tubes. After spiking the samples they were vortex mixed for 30 s. Two milliliter of solvents (acetonitrile, methanol, ethyl-acetate, dichloromethane, acetone, hexane) were added to the samples and vortex mixed again for 30 s. The mixtures were centrifuged for 15 min at 3000 rpm. Where the supernatants consisted of two separate liquid phases, the organic phase was separated and evaporated to dryness under N2 stream. The residues were reconstituted in 500 ll of acetonitrile: Milli-Q water (50:50). The samples obtained were then injected without any further purification.
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Table 1 Maximum levels, concentrations in working solutions, internal standards used and mass spectrometric parameters of the target compounds.
* **
Name
ML (lg/ kg)
Conc. in I (ng/ ml)
Conc. in II (ng/ ml)
ISTD
Parent ion
Daughter ion
Fragmentor
Collision energy
Decoquinate
20
800
–
Decoquinate-d5
418.1
200
–
Diclazuril-bis
406.8 404.8 301.0
372.1 204.0 335.9 333.9 137.0
180
**
20 45 15 15 10
Diclazuril
5
4,40 -Dinitrocarbanilide
5
200
–
4,40 -Dinitrocarbanilided8
Halofuginone
1
40
–
Robenidine-d8
415.9
Lasalocid
1
40
–
Nigericin
613.2
Maduramicin
2
80
–
Nigericin
939.5
Monensin
2*
80
–
Nigericin
693.3
Narasin
1
40
–
Nigericin
787.4
Robenidine
5
200
–
Robenidine-d8
333.9
Salinomycin
2
80
–
Nigericin
773.4
Semduramicin
2
80
–
Nigericin
895.4
Decoquinate-d5 (ISTD)
–
–
800
–
423.1
Diclazuril-bis (ISTD)
–
–
200
–
4,40 -Dinitrocarbanilide-d8 (ISTD)
–
–
200
–
420.8 418.8 309.0
Nigericin (ISTD)
–
–
80
–
747.4
Robenidine-d8 (ISTD)
–
–
200
–
342.0
107.0 138.0 121.0 577.2 377.2 895.5 877.5 675.3 461.1 531.2 431.2 155.0 138.0 531.1 431.1 851.4 833.4 377.1 205.0 349.8 347.9 141.1 111.1 703.3 501.2 159.0 142.0
80 80 80
120 140 220 160 200 100 240 160 160 80 80 80
200 100
45 15 30 35 40 55 30 40 60 50 60 20 25 55 50 35 35 25 45 20 20 10 45 60 60 20 25
MRL defined by 2377/1990/EC. Not subject to MRL in ruminants; 5 lg/kg used arbitrarily.
2.5.3. Solid phase extraction Beginning of the procedure is the same as described in Section 2.4.2. Extraction solvent was acetonitrile. After centrifugation 3 ml aliquot of the supernatant was transferred into a second centrifuge tube. 6 ml of Milli-Q water was added. The resulting samples were vortex mixed for 10 s, and then purified to remove the matrix compounds using different kinds of cartridges (Oasis HLB, 60 mg; Agilent OPT, 60 mg; Agilent C-18 (EC), 200 mg; Agilent C18 (EC), 500 mg; Strata-X, 200 mg). Prior to sample administration the cartridges had been conditioned with 3 ml acetonitrile and 3 ml Milli-Q water. After loading the whole amount of the diluted extract, the cartridges were washed with 3 ml of Milli-Q water, and subsequently dried at constant air flow for 15 min. Elution was performed with 3 ml of acetonitrile. The elutant was evaporated to dryness under N2 stream at ambient temperature. The residue was reconstituted in 500 ll of acetonitrile: Milli-Q (50:50%) and vortex mixed for 30 s prior to injection.
samples (1.5%, 2.8% and 3.5% of fat content) to the validation procedure. Quantitative analysis was carried out applying matrix-matched calibration curves. For building up the calibration curve blank milk samples were fortified with coccidiostats at five different levels in the range of 0.5 ML to 5 ML. Selectivity of the method was verified by analysing blank samples with different fat content. Sensitivity and linearity were assessed by plotting the relative peak areas of analytes versus concentrations. Calculated recoveries and within-laboratory reproducibilities were based on three days’ validation data. CCa and CCb values described in 2002/657/EC (2002) were also determined for the developed method. Limit of detection (LOD) and limit of quantification (LOQ) (not indicated in 2002/657/EC 2002) were investigated as well. Blank milk samples fortified with coccidiostats at descending order were analysed. LODs and LOQs were determined based on signal to noise ratios (LOD P 3; LOQ P 10).
2.6. Validation
3. Results and discussion
Method validation was performed according to Commission Decision 2002/657/EC (2002) with the sample preparation defined in Section 2.4.3. using Oasis HLB (60 mg) cartridges. Blank samples were fortified at the concentration of 0.5 ML, ML and 1.5 ML with six parallels over three days. Calibration samples and three blank samples were also included. Testing the robustness of the method was performed by randomly inserting different milk
3.1. Method development 3.1.1. Instrumental The ionophore coccidiostats tend to form sodium adducts. Thus for all of them the abundant [M+Na]+ ions were set as parent ions. [M+H]+ was selected for robenidine, decoquinate and halofuginone whereas [MH] for DNC and diclazuril. For unambiguous
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S. Nász et al. / Food Chemistry 133 (2012) 536–543 Table 2 Absolute recovery (recovery without internal standard correction) of analytes by SPE (%). Cartridges used
Oasis HLB (60 mg) Agilent OPT (60 mg) Agilent C18 (EC) (200 mg) Agilent C18 (EC) (500 mg) Phenomenex StrataX (200 mg)
Absolute recovery (%) Decoquinate
Diclazuril
Dinitrocarbanilide
Halofuginone
Lasalocid
Maduramicin
Monensin
Narasin
Robenidine
Salinomycin
42.0 40.6 39.3
68.0 65.1 62.9
8.9 15.1 16.0
60.3 35.4 33.4
18.9 21.7 18.1
23.2 25.4 23.0
23.9 24.9 27.2
51.2 56.9 69.5
64.3 68.3 74.2
32.5 35.4 37.1
34.3
58.1
11.6
25.0
10.2
23.6
27.6
67.4
31.8
11.7
17.4
93.6
0.3
13.8
21.4
18.6
27.3
44.3
1.9
40.3
Table 3 Absolute recovery (recovery without internal standard correction) of analytes by MSPD (%). Solvents used
Acetonitrile Methanol Ethyl-acetate Dichloromethane Acetone Hexane
Absolute recovery (%) Decoquinate
Diclazuril
Dinitrocarbanilide
Halofuginone
Lasalocid
Maduramicin
Monensin
Narasin
Robenidine
Salinomycin
1.8 31.9 11.2 8.5 8.6 0.4
52.0 38.2 41.2 45.2 27.6 0.0
40.8 21.6 41.5 57.6 26.8 0.1
5.9 5.2 0.5 0.2 4.5 0.0
1.2 21.5 26.2 3.5 8.4 0.1
1.3 51.5 67.2 22.7 27.0 0.0
4.2 44.8 54.8 15.8 33.7 0.1
6.3 17.7 13.4 1.9 17.3 0.0
4.6 14.0 14.4 4.7 18.4 0.2
7.3 49.4 58.1 27.7 39.3 0.1
identification of the analytes two transitions were set for each. Relative retention times and qualifier/quantifier ion ratios were also monitored to ensure that they are in the range accepted by 2002/ 657/EC (2002). For quantitative evaluation always the more intense transition was used. Though coccidiostats are often separated on C18 colums (Dubois et al., 2004; Mortier, Daeseleire, & Delahaut, 2003) with acidified acetonitrile and water gradients, Dubreil-Chéneau et al., 2009, as well as Cronly et al. (2010) preferred C8 stationary phase. We tested Agilent Zorbax SB C18 (4.6 75 mm, 3.5 lm) and Agilent Eclipse XDB C8 (3 150 mm, 3.5 lm). The C18 phase proved to be unsatisfactory: retention times of several compounds were too big even with eluents having high organic content, moreover, the peak shape of lasalocid was rather distorted. These problems did not occur in the case of the C8 phase. The gradient programme was optimised in order to have an efficient run time and allow successful segmentation of MRM. It provides baseline separation in 15 min for each compound with the exception of the co-eluting pair of semduramicin and lasalocid. Column temperature was varied between 30 and 60 °C and set to 30 °C, since elevating the temperature had no significant advantages. In order to further improve peak shapes methanol was added to the mobile phase (0%, 10%, and 20%). Nevertheless, when the separation was run without methanol the peaks were slightly higher. Besides, no notable changes in the peak shapes could be observed as a result of the methanol addition. Thus finally Milli-Q water and acetonitrile, both acidified with formic acid were chosen as mobile phases.
3.1.2. Sample preparation During method development for sample preparation two key points had to be kept in mind. Firstly, the low concentration levels defined in 124/2009/EC (2009) and 2377/90/EC (1990) mean that dilution of the sample regarding the whole process of sample preparation is not affordable, moreover enrichment is preferable. On the other hand, to minimise ion suppression/enhancement the amount of matrix components in the prepared sample should be as low as possible. As a first step a very fast and simple solvent extraction procedure was evaluated with acetonitrile, methanol, ethyl-acetate, dichloromethane, acetone and hexane. Two separate liquid phases
were obtained with ethyl-acetate, dichloromethane and hexane. Since acetonitrile gave the highest absolute recoveries, for further experiments this was applied as extraction solvent. However, due to the broad range of matrix compounds extracted, unstable detector responses were experienced. The application of SPE for clean up improved reproducibility considerably by removing the majority of background interferences. It also provided a four times enrichment. Absolute recoveries for the different SPE cartridges are summarised in Table 2. For several compounds Strata-X provided the lowest results, while in most cases all the other cartridges showed similar characteristics. The final choice of SPE phase was based on the absolute recoveries of halofuginone since this compound had both the lowest detector response and the lowest maximum limit. Matrix solid phase dispersion was also assessed, because it enables extraction and purification in one step. Still the time demand of the procedure was found to be comparable to that of SPE. As elution solvents acetonitrile, methanol, ethyl-acetate, dichloromethane, acetone and hexane were tested. Absolute recovery data are summarised in Table 3. Hexane – being the most apolar – gave the worst results for all compounds. None of the solvents gave reasonable absolute recovery for halofuginone, the most polar target compound. 3.1.3. Selection of internal standards To some extent application of appropriate internal standards can correct the error originating from sample preparation and thus increase the precision of the results even at trace concentrations. In the case of robenidine, decoquinate, diclazuril and 4–40 -dinitrocarbanilide, the corresponding deuterated analogues were applied. For ionophores, nigericin could be used as internal standard. For halofuginone robenidine-d8 proved to be an adequate match. 3.2. Validation Selectivity of the method is demonstrated in Fig. 1. Due to the high selectivity provided by MRM no significant peaks at the retention time of coccidiostats could be observed in any blank samples. Sensitivity and linearity were assessed using the matrix-matched calibration curves. In the range of 0.5 ML-5 ML the regression
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Fig. 1. Chromatograms corresponding to blank milk sample (a) and spiked milk sample at ML level with coccidiostats (b). Each chromatogram is presented on its own scale.
coefficients were above 0.98 for each analyte, except for narasin, lasalocid and halofuginine (>0.95).
Calculated recoveries and within-laboratory reproducibilities were calculated for three days’ data and summarised in Table 4.
S. Nász et al. / Food Chemistry 133 (2012) 536–543
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Fig. 1 (continued)
Calculated recoveries ranged between 77.1% and 118.2% for all compounds, for all tested levels. The relative standard deviations (RSD%) characterising within-laboratory reproducibilities are
acceptable for all compounds, except lasalocid. At ML values showed even better correspondence. The unacceptably high RSD%s for lasalocid are not unprecedented. Olejnik et al. (2009) suggest
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S. Nász et al. / Food Chemistry 133 (2012) 536–543
Table 4 Validation results calculated over three days. Compound name
Decoquinate Diclazuril Dinitrocarbanilide Halofuginone Lasalocid Maduramicin Monensin Narasin Robenidine Salinomycin Semduramicin *
Calculated recovery* (%)
Within laboratory reproducibility, CV (%)
0.5 ML
ML
1.5 ML
0.5 ML
ML
1.5 ML
99.7 82.6 92.5 111.3 93.2 86.9 85.0 100.7 101.1 89.8 77.1
97.7 96.6 92.3 91.6 110.8 96.3 97.6 118.2 99.7 97.7 94.8
97.0 102.5 95.9 102.6 113.9 100.7 97.0 110.4 99.7 94.6 100.5
7.17 25.22 27.16 19.74 78.97 17.21 12.08 52.73 10.55 8.47 22.33
3.48 16.10 8.23 16.33 55.14 9.11 9.68 32.67 3.87 7.68 16.77
3.14 23.72 4.43 24.99 106.84 15.05 5.65 35.91 4.83 5.57 19.67
CCa (lg/kg)
CCb (lg/kg)
LOD (lg/kg)
LOQ (lg/kg)
21.11 6.28 5.62 1.25 2.00 2.29 2.31 1.63 5.32 2.25 2.52
22.23 7.55 6.25 1.49 3.00 2.58 2.62 2.27 5.63 2.49 3.04
0.10 0.10 0.50 0.50 0.05 0.05 0.01 0.01 0.05 0.01 0.01
0.50 1.00 1.00 1.00 0.50 0.50 0.10 0.10 0.50 0.10 0.10
Recovery after internal standard correction.
that this is due to irreproducible retention on Alumina N columns, which they used for defatting chicken liver extracts before clean up on Oasis HLB cartridges. They mention that no such problem occurs in the case of egg extracts purified solely on Oasis HLB. Yet our experiments showed unambiguously that the major source of the uncertainty is the sample preparation even though no Alumina was used. Decision limit (CCa) and detection capability (CCb) as indicated in 2002/657/EC (2002) are given in Table 4. LODs and LOQs for the whole procedure were investigated experimentally, avoiding extrapolation. The results are summarised in Table 4. For most of the analytes LODs are at least one order lower than the ML values, for many of them, this applies for the LOQ values as well. The robustness of the developed method was tested by including milk samples having different fat content (1.5%, 2.8%, and 3.5%) randomly in the validation procedure. Results showed that neither calculated recoveries nor reproducibilities are affected by the fat content of the sample. 4. Conclusions Coccidiostats are widely used veterinary pharmaceuticals. With respect to the unavoidable carry-over of substances into non-target feed that may result in undesirable contaminants in derived food of animal origin, Maximum Levels were established for 11 coccidiostats in 2009 (124/2009/EC, 2009). The levels in milk vary between 1 and 20 lg/kg. Even though MSPD realises extraction and clean up simultaneously, solvent extraction coupled with SPE proved to be more advantageous. Thus the main steps of the proposed analytical method are solvent extraction with acetonitrile, SPE clean up of the extract on Oasis HLB phase, concentration by evaporation and LC–MS–MS determination. For separation of the compounds C8 was found to be superior to C18 phase. To achieve low detection limits and the necessary selectivity the LC–MS–MS was operated in MRM mode. The method was validated over three days according to 2002/ 657/EC (2002). Because of the poor reproducibility for lasalocid, the method can be used as a screening method for this compound. For all the other compounds it was found to be applicable as confirmatory method. Acknowledgements We gratefully thank Kromat Ltd for providing the LC-MS-MS instrument. We would like to acknowledge Janssen for kindly donating diclazuril-bis and Merck for providing the chemicals.
References Commission Regulation 124/2009/EC (2009). COMMISSION REGULATION (EC) No 124/2009 of 10 February 2009 setting maximum levels for the presence of coccidiostats or histomonostats in food resulting from the unavoidable carryover of these substances in non-target feed. Official Journal of the European Union, L40, 7–11. Council Regulation 2377/90/EC (1990). COUNCIL REGULATION (EEC) No 2377/90 of 26 June 1990 laying down a Community procedure for the establishment of maximum residue limits of veterinary medicinal products in foodstuffs of animal origin. Official Journal of the European Union, L224, 1–136. Cannavan, A., Ball, G., & Kennedy, G. (1999). Determination of nicarbazin in feeds using liquid chromatography–electrospray mass spectrometry. The analyst, 124, 1431–1434. Chéneau, E., Henri, J., Pirotais, Y., Abjean, J.-P., Roudaut, B., Sanders, P., et al. (2007). Liquid chromatography–electrospray tandem mass spectrometric method for quantification of monensin in plasma and edible tissues of chicken used in pharmacokinetic studies: Applying a total error approach. Journal of Chromatography B, 850, 15–23. Cronly, M., Behan, P., Foley, B., Malone, E., Shearan, P., & Regan, L. (2010). Determination of eleven coccidiostats in animal feed by liquid chromatography–tandem mass spectrometry at cross contamination levels. Analytica Chimica Acta, in press, doi:10.1016/j.aca.2010.11.001. Commission Decision 2002/657/EC (2002). Commission Decision of 12 August 2002 implementing Council Directive 96/23/EC concerning the performance of analytical methods and the interpretation of results. Official Journal of the European Communities, L221, 8–36. Dubois, M., Pierret, G., & Delahaut, Ph. (2004). Efficient and sensitive detection of residues of nine coccidiostats in egg and muscle by liquid chromatography– electrospray tandem mass spectrometry. Journal of Chromatography B, 813, 181–189. Dubreil-Chéneau, E., Bessiral, M., Roudaut, B., Verdon, E., & Sanders, P. (2009). Validation of a multi-residue liquid chromatography–tandem mass spectrometry confirmatory method for 10 anticoccidials in eggs according to Commission Decision 2002/657/EC. Journal of Chromatography A, 1216, 8149–8157. Dusi, G., Faggionato, L., Gamba, V., & Baiguera, A. (2000). Determination of nicarbazin and clopidol in poultry feeds by liquid chromatography. Journal of Chromatography A, 882, 79–84. Dusi, G., & Gamba, V. (1999). Liquid chromatography with ultraviolet detection of lasalocid, monensin, salinomycin and narasin in poultry feeds using pre-column derivatization. Journal of Chromatography A, 835, 243–246. Commission Regulation 1831/2003/EC (2003). Regulation (EC) No 1831/2003 of the European Parliament and of the Council of 22 September 2003 on additives for use in animal nutrition. Official Journal of the European Union, L268, 29–43. Feás, X., Vázquez, B. I., Iglesias, A., Cantalapiedra, J., Fente, C. A., & Cepeda, A. (2010). Sensitive determination of decoquinate in milk by high-performance liquid chromatographic coupled to laser-induced fluorescence detection. Food Analyst Methods, 3, 138–143. Huebra, M. J. G., Vincent, U., & von Holst, C. (2010). Determination of semduramicin in poultry feed at authorized level by liquid chromatography single quadrupole mass spectrometry. Journal of Pharmaceutical and Biomedical Analysis, 53, 860–868. Matabudul, D. K., Crosby, N. T., Lumley, I., & Sumara, S. (2001). The optimisation of a rapid method for the determination of lasalocid in poultry feed using supercritical fluid extraction and high performance liquid chromatography. Food Chemistry: Analytical, Nutritional and Clinical Methods Section, 75, 465–471. Mortier, L., Daeseleire, E., & Delahaut, P. (2003). Simultaneous detection of five coccidiostats in eggs by liquid chromatography–tandem mass spectrometry. Analytica Chimica Acta, 483, 27–37. Mortier, L., Daeseleire, E., & Peteghem, C. V. (2005a). Liquid chromatographic tandem mass spectrometric determination of five coccidiostats in poultry eggs and feed. Journal of Chromatography B, 820, 261–270.
S. Nász et al. / Food Chemistry 133 (2012) 536–543 Mortier, L., Daeseleire, L., & Peteghem, C. V. (2005b). Determination of the coccidiostat diclazuril in poultry feed and meat by liquid chromatography– tandem mass spectrometry. Analytica Chimica Acta, 529, 229–234. Olejnik, M., Szprengier-Juszkiewicz, T., & Jedziniak, P. (2009). Multi-residue confirmatory method for the determination of twelve coccidiostats in chicken liver using liquid chromatography tandem mass spectrometry. Journal of Chromatography A, 1216, 8141–8148. Shao, B., Wu, X., Zhang, J., Duan, H., Chu, X., & Wu, Y. (2009). Development of a Rapid LC–MS–MS Method for Multi-Class Determination of 14 Coccidiostat Residues in Eggs and Chicken. Chromatographia, 69, 1083–1088. Thompson, T. S., Noot, D. K., & Kendall, J. D. (2011). Determination of ionophores in raw bovine milk using LC–MS/MS: Application to residue surveillance. Food Chemistry, 127, 321–326.
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Vincent, U., Chedin, M., Yasar, S., & von Holst, C. (2008). Determination of ionophore coccidiostats in feedingstuffs by liquid chromatography–tandem mass spectrometry. Part I. Application to targeted feed. Journal of Pharmaceutical and Biomedical Analysis, 47, 750–757. Vincent, U., Ezerskis, Z., Chedin, M., & von Holst, C. (2011). Determination of ionophore coccidiostats in feeding stuffs by liquid chromatography-tandem mass spectrometry. Part II. Application to cross-contamination levels and nontargeted feed. Journal of Pharmaceutical and Biomedical Analysis, 54, 526–534. Yakkundi, S., Cannavan, A., Elliott, C. T., Lövgren, T., & Kennedy, D. G. (2003). D evelopment and validation of a method for the confirmation of halofuginone in chicken liver and eggs using electrospray tandem mass spectrometry. Journal of Chromatography B, 788, 29–36.
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Analytical Methods
Development and validation of a liquid chromatographic-tandem mass spectrometric method for determination of eleven coccidiostats in milk Szilárd Nász a, Lajos Debreczeni b, Tamás Rikker c, Zsuzsanna Eke a,c,⇑ a
Joint Research and Training Laboratory on Separation Techniques, Eötvös Loránd University, 1117 Budapest, Pázmány Péter Sétány 1/A, Hungary Central Agricultural Office, Food and Feed Safety Directorate, Feed Investigation National Reference Laboratory, 1144 Budapest, Remény utca 42, Hungary c Wessling International Research and Educational Center, 1047 Budapest, Fóti út 56, Hungary b
a r t i c l e
i n f o
Article history: Received 16 March 2011 Received in revised form 12 October 2011 Accepted 14 January 2012 Available online 25 January 2012 Keywords: LC–MS–MS Coccidiostats Milk Validation Solid phase extraction
a b s t r a c t A reversed phase liquid chromatographic–tandem mass spectrometric method with simple solvent extraction and purification by solid phase extraction (SPE) has been developed for the determination of coccidiostats in milk. For sample preparation matrix solid phase dispersion, extraction by organic solvent and SPE with different cartridges were also tested. The compounds determined include lasalocid, narasin, salinomycin, monensin, semduramicin, maduramicin, robenidine, decoquinate, halofuginone, nicarbazin and diclazuril. Main steps of the method are addition of acetonitrile to the milk samples, centrifugation, removal of matrix by SPE, concentration by evaporation and LC–MS–MS determination. During a 15 min time segmented chromatographic run compounds are ionised either positively or negatively. Calculated recoveries range between 77.1% and 118.2%. Maximum levels are in the range of 1–20 lg/kg. The developed method was validated in line with the requirements of Commission Decision 2002/657/EC (2002). It is applicable for control of coccidiostat residues in milk as indicated in Regulation 124/2009/EC (2009). Ó 2012 Elsevier Ltd. All rights reserved.
1. Introduction Coccidiostats are widely used veterinary pharmaceuticals intended to kill or inhibit protozoa. The usage of anticoccidial drugs as feed additives was licensed by the European Union in 2003 (1831/2003/EC, 2003) to allow for the prevention of coccidiosis, a disease that may cause serious economical consequences. Rules of utilisation are specified for different groups of animals. Nevertheless, the so-called unavoidable carry-over of substances into non-target feed may result in undesirable contaminants in derived food of animal origin. Hence maximum levels (ML) were established for 11 coccidiostats in 2009 (124/2009/EC, 2009). Coccidiostats can be classified into two groups. Robenidine, decoquinate, halofuginone, nicarbazin and diclazuril are chemically produced drugs. Lasalocid, narasin, salinomycin, monensin, semduramicin and maduramicin are ionophores. Their chemical structure and polarity varies greatly, particularly in the group of the chemically produced ones. Furthermore nicarbazin is an equimolar complex of 1,3-bis(4-nitrophenyl)urea and 4,6dimethyl-1H-pyrimidin-2-one. These compounds are also known ⇑ Corresponding author at: Joint Research and Training Laboratory on Separation Techniques, Eötvös Loránd University, 1117 Budapest, Pázmány Péter Sétány 1/A, Hungary. Tel.: +36 30 598 0300. E-mail addresses: [email protected] (S. Nász), [email protected] (Z. Eke). 0308-8146/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved. doi:10.1016/j.foodchem.2012.01.022
as 4,40 -dinitrocarbanilide (DNC) and 2-hydroxy-4,6-dimethylpyrimidine (DHP), respectively. DHP excretes fast therefore it is less likely to appear in any food of animal origin. Thus it is reasonable to measure only DNC as a representative of nicarbazin. Several papers describe methods for the determination of coccidiostats. Due to the high boiling point of most coccidiostats liquid chromatography is preferred, usually coupled to tandem mass spectrometry (LC–MS–MS) (Shao et al., 2009; Thompson, Noot, & Kendall, in press; Vincent, Ezerskis, Chedin, & von Holst, 2011; Yakkundi, Cannavan, Elliott, Lövgren, & Kennedy, 2003). UV (Dusi & Gamba, 1999), fluorescence (Matabudul, Crosby, Lumley, & Sumara, 2001) and single quadrupole mass spectrometric detection (Huebra, Vincent, & von Holst, 2010) may also be suitable. Many of these methods are applicable for only a few coccidiostats. Mortier, Daeseleire, and Peteghem (2005a) described a method for five chemical coccididiostats and one metabolite in poultry eggs and feed. Dusi and Gamba (1999) determined four ionophore compounds in poultry feeds, whereas Vincent, Chedin, Yasar, and von Holst (2008) developed a method for six ionophores in poultry and cattle compound feed. Methods for individual compounds like halofuginone (Yakkundi et al., 2003), decoquinate (Feás et al., 2010), diclazuril (Mortier, Daeseleire, & Peteghem, 2005b), nicarbazin (Cannavan, Ball, & Kennedy, 1999; Dusi, Faggionato, Gamba, & Baiguera, 2000), lasalocid (Matabudul et al., 2001), monensin (Chéneau et al., 2007) and semduramicin (Huebra et al., 2010) in different matrices are described as well. In contrast to that only a
S. Nász et al. / Food Chemistry 133 (2012) 536–543
few multi-class methods have been developed for coccidiostats (Cronly et al. 2010; Dubois, Pierret, & Delahaut, 2004; Dubreil-Chéneau, Bessiral, Roudaut, Verdon, & Sanders 2009; Olejnik, Szprengier-Juszkiewicz, & Jedziniak, 2009) and to our knowledge none for milk samples. The maximum levels and maximum residue limits (MRL) for milk given in 124/2009/EC (2009) and 2377/90/EC (1990) are in the range of 1–20 lg/kg except for diclazuril. Since according to 2377/90/EC (1990) this compound is not subject to MRL in ruminants, a reference concentration of 5 lg/kg was used arbitrarily throughout this study. Determination of residues at this concentration level requires the most selective and sensitive detection. Thus triple quadruple detection and multiple reaction monitoring (MRM) are indispensable. For sample preparation matrix solid phase dispersion (MSPD), extraction by organic solvent without any further purification and solid phase extraction (SPE) with different cartridges have also been tested. The developed method is fully validated according to 2002/657/EC (2002) and allows the determination of coccidiostats at trace level. 2. Materials and methods 2.1. Reagents and chemicals Narasin, maduramicin, decoquinate, DNC, DNC-d8, robenidined8, decoquinate-d5 and nigericin were purchased from Sigma (Budapest, Hungary). Lasalocid, salinomycin, monensin, robenidine, halofuginone and diclazuril were purchased from LGC (Wesel, Germany) and semduramicin from Phibro (Ridgefield Park, USA). Diclazuril-bis was kindly donated by Janssen (Beerse, Belgium). Formic acid (98–100%) was from Merck (Budapest, Hungary). For matrix solid phase dispersion Isolute C18 (EC) and for solid phase extraction Oasis HLB (60 mg), Agilent OPT (60 mg), Agilent C-18 (EC) (200 mg), Agilent C-18 (EC) (500 mg), Strata-X (200 mg) were used. All the solvents used were of analytical grade. Water was purified by Milli-Q system (Milli-Q, USA). Analytical grade sodium-formiate was from Reanal (Budapest, Hungary). Stock solutions, containing 1 mg/ml of coccidiostats, were prepared individually. Maduramicin, narasin, nigericin, monensin, salinomycin, and semduramicin were dissolved in methanol, DNC, DNC-d8, diclazuril, diclazuril-bis, robenidine, robenidine-d8 in dimethylsulfoxid (DMSO), decoquinate and decoquinate-d5 in acetonitrile with 6% formic acid. Halofuginone and lasalocid were purchased as 100 lg/ml solutions in acetonitrile. Working solutions were prepared by diluting the individual stock solutions with acetonitrile. Table 1 shows the concentrations of mixed working solution I (target compounds) and II (ISTDs). 2.2. Milk samples For development and validation purposes bovine milk samples with different fat content (1.5%, 2.8%, 3.5%) were obtained from Hungarian retail markets. No residues of target analytes were found in these samples either by the analysis of acetonitrile extract of milk samples or by preparation of milk samples according to the presented procedure thus they were used as blank matrices. 2.3. Instrumentation Agilent 1200 series HPLC consisting of a degasser (G1379B), binary pump (G1312A), injector (G1367B), thermostat (G1330B) and column thermostat compartment (G1316A) coupled to Agilent 6460 triple quadrupole mass spectrometer (G6460A) equipped with Agilent Jet Stream electrospray ion source was used. The instrument was controlled and the results were evaluated by
537
MassHunter software. Hermile (Z230A) system was used for centrifugation and a Barkley (Germany) device for evaporation. Visiprep (Supelco) was used for solid phase extraction. 2.4. Chromatographic conditions Separation was achieved using an Agilent Eclipse XDB-C8 (3 150, 3.5 lm) analytical column protected by a Zorbax Eclipse Plus C8 (2.1 12.5 mm, 5 lm) guard column. The oven temperature was set at 30 °C. Eluent A was Milli-Q water, B was acetonitrile, both acidified with 0.1% formic acid. The flow rate was 0.5 ml/min with the following gradient programme: 5% acetonitrile for 1 min, then linear increase of acetonitrile to 95% over 3 min, finally holding on for 11 min. Five minute equilibration time was applied between runs. The injection volume was 30 ll with needle wash in acetonitrile in order to avoid cross-contamination. 2.4.1. Mass spectrometric parameters Jet Stream source parameters were optimised to give the most abundant ion responses for the analytes. Gas temperature was set to 350 °C, gas flow to 10 l/min, nebulizer to 40 psi, sheath gas temperature to 350 °C, sheath gas flow to 7 l/min, capillary voltages, positive and negative alike to 3500 V and nozzle voltages to 1000 V. Multiple reaction monitoring was used for quantification of the compounds because this methodology is the most selective and sensitive detection when applying triple quadrupole mass spectrometry. Individual standard solutions (1 lg/ml in acetonitrile: Milli-Q 50:50) were injected to optimise the transition parameters. As required by the Commission Decision 2002/657/ EC (2002) two transitions were chosen for each coccidiostats. Table 1 shows the chosen transitions as well as the optimised fragmentor voltages and collision energies. The chromatographic run was divided into three time segments: from 0 to 6.3 min positive ion mode, in the second segment from 6.3 to 7.1 min negative ion mode and in the third segment from 7.1 to 15 min positive ion mode was applied. 2.5. Sample preparation 2.5.1. Matrix solid phase dispersion 2 g MSPD sorbent was placed in a mortar and 0.5 ml milk sample spiked at 100 ng/ml was added. It was homogenised with a pestle for 30 s. A syringe barrel containing a filter at the bottom was filled in with the mixture. Six parallels were prepared and the compounds were eluted with different solvents: acetonitrile, methanol, ethyl-acetate, dichloromethane, acetone and hexane. The volume of the solvents was 8 ml in each case. Elutants were collected in glass vials and evaporated to dryness under continuous N2 stream at ambient temperature. The residues were then reconstituted in 500 ll of acetonitrile: Milli-Q water (50:50) and vortex mixed for 30 s prior to injection. 2.5.2. Solvent extraction Two milliliter aliquots of milk samples were placed in centrifuge tubes. After spiking the samples they were vortex mixed for 30 s. Two milliliter of solvents (acetonitrile, methanol, ethyl-acetate, dichloromethane, acetone, hexane) were added to the samples and vortex mixed again for 30 s. The mixtures were centrifuged for 15 min at 3000 rpm. Where the supernatants consisted of two separate liquid phases, the organic phase was separated and evaporated to dryness under N2 stream. The residues were reconstituted in 500 ll of acetonitrile: Milli-Q water (50:50). The samples obtained were then injected without any further purification.
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Table 1 Maximum levels, concentrations in working solutions, internal standards used and mass spectrometric parameters of the target compounds.
* **
Name
ML (lg/ kg)
Conc. in I (ng/ ml)
Conc. in II (ng/ ml)
ISTD
Parent ion
Daughter ion
Fragmentor
Collision energy
Decoquinate
20
800
–
Decoquinate-d5
418.1
200
–
Diclazuril-bis
406.8 404.8 301.0
372.1 204.0 335.9 333.9 137.0
180
**
20 45 15 15 10
Diclazuril
5
4,40 -Dinitrocarbanilide
5
200
–
4,40 -Dinitrocarbanilided8
Halofuginone
1
40
–
Robenidine-d8
415.9
Lasalocid
1
40
–
Nigericin
613.2
Maduramicin
2
80
–
Nigericin
939.5
Monensin
2*
80
–
Nigericin
693.3
Narasin
1
40
–
Nigericin
787.4
Robenidine
5
200
–
Robenidine-d8
333.9
Salinomycin
2
80
–
Nigericin
773.4
Semduramicin
2
80
–
Nigericin
895.4
Decoquinate-d5 (ISTD)
–
–
800
–
423.1
Diclazuril-bis (ISTD)
–
–
200
–
4,40 -Dinitrocarbanilide-d8 (ISTD)
–
–
200
–
420.8 418.8 309.0
Nigericin (ISTD)
–
–
80
–
747.4
Robenidine-d8 (ISTD)
–
–
200
–
342.0
107.0 138.0 121.0 577.2 377.2 895.5 877.5 675.3 461.1 531.2 431.2 155.0 138.0 531.1 431.1 851.4 833.4 377.1 205.0 349.8 347.9 141.1 111.1 703.3 501.2 159.0 142.0
80 80 80
120 140 220 160 200 100 240 160 160 80 80 80
200 100
45 15 30 35 40 55 30 40 60 50 60 20 25 55 50 35 35 25 45 20 20 10 45 60 60 20 25
MRL defined by 2377/1990/EC. Not subject to MRL in ruminants; 5 lg/kg used arbitrarily.
2.5.3. Solid phase extraction Beginning of the procedure is the same as described in Section 2.4.2. Extraction solvent was acetonitrile. After centrifugation 3 ml aliquot of the supernatant was transferred into a second centrifuge tube. 6 ml of Milli-Q water was added. The resulting samples were vortex mixed for 10 s, and then purified to remove the matrix compounds using different kinds of cartridges (Oasis HLB, 60 mg; Agilent OPT, 60 mg; Agilent C-18 (EC), 200 mg; Agilent C18 (EC), 500 mg; Strata-X, 200 mg). Prior to sample administration the cartridges had been conditioned with 3 ml acetonitrile and 3 ml Milli-Q water. After loading the whole amount of the diluted extract, the cartridges were washed with 3 ml of Milli-Q water, and subsequently dried at constant air flow for 15 min. Elution was performed with 3 ml of acetonitrile. The elutant was evaporated to dryness under N2 stream at ambient temperature. The residue was reconstituted in 500 ll of acetonitrile: Milli-Q (50:50%) and vortex mixed for 30 s prior to injection.
samples (1.5%, 2.8% and 3.5% of fat content) to the validation procedure. Quantitative analysis was carried out applying matrix-matched calibration curves. For building up the calibration curve blank milk samples were fortified with coccidiostats at five different levels in the range of 0.5 ML to 5 ML. Selectivity of the method was verified by analysing blank samples with different fat content. Sensitivity and linearity were assessed by plotting the relative peak areas of analytes versus concentrations. Calculated recoveries and within-laboratory reproducibilities were based on three days’ validation data. CCa and CCb values described in 2002/657/EC (2002) were also determined for the developed method. Limit of detection (LOD) and limit of quantification (LOQ) (not indicated in 2002/657/EC 2002) were investigated as well. Blank milk samples fortified with coccidiostats at descending order were analysed. LODs and LOQs were determined based on signal to noise ratios (LOD P 3; LOQ P 10).
2.6. Validation
3. Results and discussion
Method validation was performed according to Commission Decision 2002/657/EC (2002) with the sample preparation defined in Section 2.4.3. using Oasis HLB (60 mg) cartridges. Blank samples were fortified at the concentration of 0.5 ML, ML and 1.5 ML with six parallels over three days. Calibration samples and three blank samples were also included. Testing the robustness of the method was performed by randomly inserting different milk
3.1. Method development 3.1.1. Instrumental The ionophore coccidiostats tend to form sodium adducts. Thus for all of them the abundant [M+Na]+ ions were set as parent ions. [M+H]+ was selected for robenidine, decoquinate and halofuginone whereas [MH] for DNC and diclazuril. For unambiguous
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S. Nász et al. / Food Chemistry 133 (2012) 536–543 Table 2 Absolute recovery (recovery without internal standard correction) of analytes by SPE (%). Cartridges used
Oasis HLB (60 mg) Agilent OPT (60 mg) Agilent C18 (EC) (200 mg) Agilent C18 (EC) (500 mg) Phenomenex StrataX (200 mg)
Absolute recovery (%) Decoquinate
Diclazuril
Dinitrocarbanilide
Halofuginone
Lasalocid
Maduramicin
Monensin
Narasin
Robenidine
Salinomycin
42.0 40.6 39.3
68.0 65.1 62.9
8.9 15.1 16.0
60.3 35.4 33.4
18.9 21.7 18.1
23.2 25.4 23.0
23.9 24.9 27.2
51.2 56.9 69.5
64.3 68.3 74.2
32.5 35.4 37.1
34.3
58.1
11.6
25.0
10.2
23.6
27.6
67.4
31.8
11.7
17.4
93.6
0.3
13.8
21.4
18.6
27.3
44.3
1.9
40.3
Table 3 Absolute recovery (recovery without internal standard correction) of analytes by MSPD (%). Solvents used
Acetonitrile Methanol Ethyl-acetate Dichloromethane Acetone Hexane
Absolute recovery (%) Decoquinate
Diclazuril
Dinitrocarbanilide
Halofuginone
Lasalocid
Maduramicin
Monensin
Narasin
Robenidine
Salinomycin
1.8 31.9 11.2 8.5 8.6 0.4
52.0 38.2 41.2 45.2 27.6 0.0
40.8 21.6 41.5 57.6 26.8 0.1
5.9 5.2 0.5 0.2 4.5 0.0
1.2 21.5 26.2 3.5 8.4 0.1
1.3 51.5 67.2 22.7 27.0 0.0
4.2 44.8 54.8 15.8 33.7 0.1
6.3 17.7 13.4 1.9 17.3 0.0
4.6 14.0 14.4 4.7 18.4 0.2
7.3 49.4 58.1 27.7 39.3 0.1
identification of the analytes two transitions were set for each. Relative retention times and qualifier/quantifier ion ratios were also monitored to ensure that they are in the range accepted by 2002/ 657/EC (2002). For quantitative evaluation always the more intense transition was used. Though coccidiostats are often separated on C18 colums (Dubois et al., 2004; Mortier, Daeseleire, & Delahaut, 2003) with acidified acetonitrile and water gradients, Dubreil-Chéneau et al., 2009, as well as Cronly et al. (2010) preferred C8 stationary phase. We tested Agilent Zorbax SB C18 (4.6 75 mm, 3.5 lm) and Agilent Eclipse XDB C8 (3 150 mm, 3.5 lm). The C18 phase proved to be unsatisfactory: retention times of several compounds were too big even with eluents having high organic content, moreover, the peak shape of lasalocid was rather distorted. These problems did not occur in the case of the C8 phase. The gradient programme was optimised in order to have an efficient run time and allow successful segmentation of MRM. It provides baseline separation in 15 min for each compound with the exception of the co-eluting pair of semduramicin and lasalocid. Column temperature was varied between 30 and 60 °C and set to 30 °C, since elevating the temperature had no significant advantages. In order to further improve peak shapes methanol was added to the mobile phase (0%, 10%, and 20%). Nevertheless, when the separation was run without methanol the peaks were slightly higher. Besides, no notable changes in the peak shapes could be observed as a result of the methanol addition. Thus finally Milli-Q water and acetonitrile, both acidified with formic acid were chosen as mobile phases.
3.1.2. Sample preparation During method development for sample preparation two key points had to be kept in mind. Firstly, the low concentration levels defined in 124/2009/EC (2009) and 2377/90/EC (1990) mean that dilution of the sample regarding the whole process of sample preparation is not affordable, moreover enrichment is preferable. On the other hand, to minimise ion suppression/enhancement the amount of matrix components in the prepared sample should be as low as possible. As a first step a very fast and simple solvent extraction procedure was evaluated with acetonitrile, methanol, ethyl-acetate, dichloromethane, acetone and hexane. Two separate liquid phases
were obtained with ethyl-acetate, dichloromethane and hexane. Since acetonitrile gave the highest absolute recoveries, for further experiments this was applied as extraction solvent. However, due to the broad range of matrix compounds extracted, unstable detector responses were experienced. The application of SPE for clean up improved reproducibility considerably by removing the majority of background interferences. It also provided a four times enrichment. Absolute recoveries for the different SPE cartridges are summarised in Table 2. For several compounds Strata-X provided the lowest results, while in most cases all the other cartridges showed similar characteristics. The final choice of SPE phase was based on the absolute recoveries of halofuginone since this compound had both the lowest detector response and the lowest maximum limit. Matrix solid phase dispersion was also assessed, because it enables extraction and purification in one step. Still the time demand of the procedure was found to be comparable to that of SPE. As elution solvents acetonitrile, methanol, ethyl-acetate, dichloromethane, acetone and hexane were tested. Absolute recovery data are summarised in Table 3. Hexane – being the most apolar – gave the worst results for all compounds. None of the solvents gave reasonable absolute recovery for halofuginone, the most polar target compound. 3.1.3. Selection of internal standards To some extent application of appropriate internal standards can correct the error originating from sample preparation and thus increase the precision of the results even at trace concentrations. In the case of robenidine, decoquinate, diclazuril and 4–40 -dinitrocarbanilide, the corresponding deuterated analogues were applied. For ionophores, nigericin could be used as internal standard. For halofuginone robenidine-d8 proved to be an adequate match. 3.2. Validation Selectivity of the method is demonstrated in Fig. 1. Due to the high selectivity provided by MRM no significant peaks at the retention time of coccidiostats could be observed in any blank samples. Sensitivity and linearity were assessed using the matrix-matched calibration curves. In the range of 0.5 ML-5 ML the regression
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Fig. 1. Chromatograms corresponding to blank milk sample (a) and spiked milk sample at ML level with coccidiostats (b). Each chromatogram is presented on its own scale.
coefficients were above 0.98 for each analyte, except for narasin, lasalocid and halofuginine (>0.95).
Calculated recoveries and within-laboratory reproducibilities were calculated for three days’ data and summarised in Table 4.
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Fig. 1 (continued)
Calculated recoveries ranged between 77.1% and 118.2% for all compounds, for all tested levels. The relative standard deviations (RSD%) characterising within-laboratory reproducibilities are
acceptable for all compounds, except lasalocid. At ML values showed even better correspondence. The unacceptably high RSD%s for lasalocid are not unprecedented. Olejnik et al. (2009) suggest
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Table 4 Validation results calculated over three days. Compound name
Decoquinate Diclazuril Dinitrocarbanilide Halofuginone Lasalocid Maduramicin Monensin Narasin Robenidine Salinomycin Semduramicin *
Calculated recovery* (%)
Within laboratory reproducibility, CV (%)
0.5 ML
ML
1.5 ML
0.5 ML
ML
1.5 ML
99.7 82.6 92.5 111.3 93.2 86.9 85.0 100.7 101.1 89.8 77.1
97.7 96.6 92.3 91.6 110.8 96.3 97.6 118.2 99.7 97.7 94.8
97.0 102.5 95.9 102.6 113.9 100.7 97.0 110.4 99.7 94.6 100.5
7.17 25.22 27.16 19.74 78.97 17.21 12.08 52.73 10.55 8.47 22.33
3.48 16.10 8.23 16.33 55.14 9.11 9.68 32.67 3.87 7.68 16.77
3.14 23.72 4.43 24.99 106.84 15.05 5.65 35.91 4.83 5.57 19.67
CCa (lg/kg)
CCb (lg/kg)
LOD (lg/kg)
LOQ (lg/kg)
21.11 6.28 5.62 1.25 2.00 2.29 2.31 1.63 5.32 2.25 2.52
22.23 7.55 6.25 1.49 3.00 2.58 2.62 2.27 5.63 2.49 3.04
0.10 0.10 0.50 0.50 0.05 0.05 0.01 0.01 0.05 0.01 0.01
0.50 1.00 1.00 1.00 0.50 0.50 0.10 0.10 0.50 0.10 0.10
Recovery after internal standard correction.
that this is due to irreproducible retention on Alumina N columns, which they used for defatting chicken liver extracts before clean up on Oasis HLB cartridges. They mention that no such problem occurs in the case of egg extracts purified solely on Oasis HLB. Yet our experiments showed unambiguously that the major source of the uncertainty is the sample preparation even though no Alumina was used. Decision limit (CCa) and detection capability (CCb) as indicated in 2002/657/EC (2002) are given in Table 4. LODs and LOQs for the whole procedure were investigated experimentally, avoiding extrapolation. The results are summarised in Table 4. For most of the analytes LODs are at least one order lower than the ML values, for many of them, this applies for the LOQ values as well. The robustness of the developed method was tested by including milk samples having different fat content (1.5%, 2.8%, and 3.5%) randomly in the validation procedure. Results showed that neither calculated recoveries nor reproducibilities are affected by the fat content of the sample. 4. Conclusions Coccidiostats are widely used veterinary pharmaceuticals. With respect to the unavoidable carry-over of substances into non-target feed that may result in undesirable contaminants in derived food of animal origin, Maximum Levels were established for 11 coccidiostats in 2009 (124/2009/EC, 2009). The levels in milk vary between 1 and 20 lg/kg. Even though MSPD realises extraction and clean up simultaneously, solvent extraction coupled with SPE proved to be more advantageous. Thus the main steps of the proposed analytical method are solvent extraction with acetonitrile, SPE clean up of the extract on Oasis HLB phase, concentration by evaporation and LC–MS–MS determination. For separation of the compounds C8 was found to be superior to C18 phase. To achieve low detection limits and the necessary selectivity the LC–MS–MS was operated in MRM mode. The method was validated over three days according to 2002/ 657/EC (2002). Because of the poor reproducibility for lasalocid, the method can be used as a screening method for this compound. For all the other compounds it was found to be applicable as confirmatory method. Acknowledgements We gratefully thank Kromat Ltd for providing the LC-MS-MS instrument. We would like to acknowledge Janssen for kindly donating diclazuril-bis and Merck for providing the chemicals.
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