Research Article The Determination of Six Ionophore Coccidiostats …downloads.hindawi.com/journals/tswj/2013/763402.pdf · 2019-07-31 · paper is based on the derivatisation approach

Hindawi Publishing CorporationThe Scientific World JournalVolume 2013, Article ID 763402, 8 pageshttp://dx.doi.org/10.1155/2013/763402

Research ArticleThe Determination of Six Ionophore Coccidiostats in Feed byLiquid Chromatography with Postcolumn Derivatisation andSpectrofotometric/Fluorescence Detection

MaBgorzata Olejnik, Piotr Jedziniak, and Teresa Szprengier-Juszkiewicz

Department of Pharmacology and Toxicology, National Veterinary Research Institute, Al. Partyzantow 57, 24-100 Pulawy, Poland

Correspondence should be addressed to Małgorzata Olejnik; [email protected]

Received 5 August 2013; Accepted 12 September 2013

Academic Editors: Z. Guo and D. M. Milojkovic-Opsenica

Copyright © 2013 Małgorzata Olejnik et al. This is an open access article distributed under the Creative Commons AttributionLicense, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properlycited.

The control of levels of anticoccidial feed additives in targeted feeds plays an important role in the assurance of efficiency ofanimal treatment, prevention of drug resistance, and food safety. The robust and labour-efficient method for the simultaneousdetermination of six ionophore coccidiostats (lasalocid, maduramicin, monensin, narasin, salinomycin, and semduramicin) intargeted feed has been developed. Properly grinded and homogenized feed sample was spiked with internal standard (monesinmethyl ester) and extracted with methanol. The extract was analysed with reversed phase HPLC without any further purification.The separation of the analytes with conventional C18 and core-shell columns was compared. Lasalocid was analysed withfluorescence detection, whereas other ionophores were detected with UV-Vis detector after derivatisation with vanillin in thepresence of sulfuric acid. Fortified samples and targeted feeds at authorized levels were used for method validation. Recoverywas in the range of 85–110%, depending on the analyte. The within-laboratory reproducibility did not exceed the target value fromHorwitz equation.The results of the proficiency tests (z-scores in the range of −1.0 to 1.9) confirmed the reliability of the developedprotocol.

1. Introduction

The intensive modern husbandry practices increase the rateof coccidiosis hence causing large economical losses inpoultry production. At the moment, the use of anticoccidialfeed additives is thought to be the most effective way ofthe control of this disease. In the European Union, 11coccidiostats are authorized as feed additives for poultry andrabbits.The specific documents describe individually for eachanticoccidial the target species, applied doses, and conditionsof administration, including, when necessary, withdrawaltimes [1].

Among authorized anticoccidial feed additives, iono-phore antibiotics (lasalocid, maduramicin, monensin, nara-sin, salinomycin, and semduramicin) play an essential role(Figure 1). They are used most frequently, due to their lowcost and high efficiency. The widespread use of ionophoreson the farms can promote the resistant strains of protozoa,

resulting in the diminished prophylactic efficiency of coccid-iostats; their wide use poses a risk also to human health dueto the potential occurrence of residues.The therapeutic indexof these coccidiostats is very low, and even target species maybe intoxicated when they are exposed to high levels of thosecompounds.

In conclusion, to ensure safe and effective use ofionophores, the proper concentrations, precisely as statedin authorization documents, have to be applied. Therefore,the availability of analytical methodology for robust andreliable quantification of ionophores in animal feeding stuffsis essential for both official control andmanufacturers’ qualitycontrol laboratories.

A number of methods for the determination ofionophores in animal feed have been described. Becauseof the lack of chromophoric groups in their molecules, thederivatisation step is required when spectrophotometricdetection is applied [2, 3]. Most of the available methods

2 The Scientific World Journal

H3C

H3C H3C

H3C

H3C

H3C

H3C

H3CCH3

CH3

CH3

O O

O

O

O

O

O

O

O

O

O O

O

O−

OH

HO

NH4+

Maduramicin ammoniumLasalocid sodiumH3C

H3C

H3C

CH3

CH3

CH3 CH3

CH3

O

O

O

O

O−

OH

HO

HO

Monensin sodium

H3C

H3C

H3CH3C

H3C

H3C

H3C

CH3

CH3

CH3 CH3 CH3

CH3

CH3

CH3

CH3 CH3CH3

CH3

CH3

CH3

CH3O−

O−

O−

OH

OH

OH

OH

OH

OH

OH

O

OO

O O

O

O

Na+

Na+

Na+

Na+

O O

O

O

O

O

O

H3C

H3C

H3C

H3C

H3C

H3C

H3C

H3C

H3C

H3C

H3C

H3C

CH3 CH3 CH3

CH3

CH3

CH3

OH

OH

HO

HO

HO

HO

O−

O O

O

O

O

O

O

Narasin

Salinomycin sodium

O

O

OO

O

OO

O

OO

O

Semduramicin sodium

Figure 1: Chemical structures of ionophore coccidiostats.

The Scientific World Journal 3

use postcolumn derivatisation with vanillin [4] or dimethyl-aminobenzaldehyde, DMAB [5]; such methods are sensitiveand robust and require little sample preparation.

Lately, also the mass spectrometric detection has beenapplied in the determination of coccidiostats in feed [6].AlthoughMS-basedmethods can be both used for the reliabledetermination of single representative of ionophore family[7, 8] and to screen all authorized polyether antibiotics [9],this approach is not always available to routine laboratoriesbecause of high cost of purchase and maintenance of massspectrometer. Also relatively high variation of results isobserved due to lack of labelled internal standards.

Some of the methods developed so far, using both post-column derivatisation/spectrophotometric detection andmass spectrometric approach, have been validated by inter-laboratory comparisons and harmonized as internationalnorms [10–13]. Most of the routine laboratories performinganalyses of feed stuffs for ionophore antibiotics follow theseofficial documents. Nevertheless, none of the up-to-datemethods enables the determination of all ionophores in singleanalytical run.

The multianalyte protocols are more practical for theorganization of laboratory work in terms of quality assur-ance and sample throughput. Therefore, we have decidedto develop single protocol for the determination of allauthorized ionophore coccidiostats based on the postcolumnderivatisation methodology, already successfully applied inthe determination of monensin, narasin, and salinomycin[11].

2. Material and Methods

2.1. Chemicals and Materials. Maduramicin ammonium(MAD), monensin sodium (MON), narasin from Strepto-myces auriofaciens (NAR), and salinomycin sodium (SAL)were purchased from Sigma (Germany). Lasalocid A sodium(LAS) was obtained fromDr. Ehrenstorfer. Monensin methylester (MON-ME) and semduramicin sodium (SMD) weredonated by EU-RL in Berlin.

Methanol, HPLC grade, was purchased from JT Baker(Germany). Methanol p.a., potassium hydroxide, and sul-phuric acid (98%) were obtained from POCh (Poland),vanillin from Merck (Germany), and dimethylsulfoxide(DMSO) and potassium dihydrophosphate from Sigma (Ger-many). Ultrapure water (resistance > 18mΩ) was obtainedfromMilli-Q system (Millipore, France).

2.2. Standard Solutions. Stock standard solutions (1mg/mL)were prepared by weighing 25.0mg of each reference stan-dard and dissolving in 25mLmethanol.These solutions werekept in the temperature below −18∘C for 12 months. Mixedworking standard solution (20𝜇g/mL MAD and 100 𝜇g/mLof remaining ionophores) was stored in 6–10∘C up to threemonths.

2.3. Sample Treatment. For the method developmentand validation, animal feed samples without ionophorecoccidiostats were used. The feed was ground with rotor mill

ZM200 (Retsch, Germany) and sieved through 0.5mm sieve.The sample (5 g± 0.01 g) was weighted into the polypropylenecentrifuge tube. The appropriate amount of mixed standardsolution was added to the spiked samples, and the samplewas let to stand for at least one hour. Methanol was added tothe sample so that the total solvent volume was 25mL (thevolume of methanol added with the standard solution wassubtracted), and the sample was shaken for 30 minutes at 200cycles/min (MaxQ 2000 Orbital Shaker, Thermo Scientific,USA). The sample was then centrifuged (3500 rpm, 10min),and 0.5mL of supernatant was transferred to a glass tube.Internal standard (monensin methyl ester, 10 𝜇L of 10 𝜇g/mLsolution) and 0.5mL DMSOwere added, and the sample wasevaporated under the stream of nitrogen (45∘C). The extractin DMSOwas transferred into a vial and analysed with liquidchromatography.

2.4. Instrumental Analysis. The instrumental analysis of coc-cidiostats was performed using Varian Prostar HPLC systemequipped with quaternary pump, autosampler, column oven,postcolumn derivatisation module, and two detectors—fluorescence and UV-Vis, controlled by Galaxie Workstationsoftware. Chromatographic separation of compounds wasperformed on Kinetex C18 column (150 × 4.6mm, 2.6 𝜇m,Phenomenex, USA) connected with precolumn (4 × 3mm,SecurityGuard, Phenomenex,USA).The isocratic elutionwasapplied, with mobile phase consisting of 88% methanol and12% 0.02M KH

2PO4, adjusted to pH 7.0 at 0.7mL/min flow

rate. Column oven temperature was controlled at 32∘C. AfterHPLC separation, eluate was transferred through fluores-cence detector (excitation and emission wavelength 310 and420 nm, resp.) for the lasalocid detection. Next, the eluate waspassed through the reaction cell (1.4mL) of the postcolumnderivatisation reactor (Pinnacle PCX, Pickering, USA). Thederivatisation was performed with 6% vanillin solution inmethanol (0.35mL/min) in the presence of 4% sulfuric acidin methanol (0.35mL/min), and the coil was maintained at110∘C. The derivatives of ionophores were then detected at 𝜆520 nm.The injection volume was 50 𝜇L.

2.5. Validation

2.5.1. Linearity. Standard calibration curves were prepared bythe injection of mixed standard solutions on five concentra-tion levels and plotting recorded peak areas of each analyteversus their concentrations. The equations and regressioncoefficients of the curves were calculated. Linearity of cali-bration curves was demonstrated with the F-test lack of fit,and the working range was established.

2.5.2. Sensitivity and Selectivity. The selectivity of themethodwas verified by the analysis of 10 different feed samples(intended for poultry, bovine, and swine). The limit ofdetection was calculated from the chromatograms of blanksamples based on signal-to-noise ratio (S/N value of 3). Thelimit of quantification (LOQ) was assumed to be at the lowestlevel of calibration curve; therefore, the repeatability at thislevel was verified by the analysis of six spiked poultry feedsamples.


2.6. Recovery and Precision. Blank poultry feed sampleswere spiked with coccidiostats on three different levels closeto the target concentrations specified in the authorisationdocuments. The spiking levels were 5, 10 and 20mg/kg formaduramicin and 25, 50 and 100mg/kg for other ionophores.

For the repeatability study, three series were analysed(six samples for each spiking level). Standard deviation (SD)and coefficient of variation (CV, %) were calculated for eachlevel. The within-laboratory reproducibility was obtained byanalysis of two additional series (on all three levels) in thereproducibility conditions (two different occasions, anothertechnician), and overall SD and CV were calculated.

The coefficient of variation of intralaboratory repro-ducibility (𝑛 = 18) was compared with the target deviationaccording to the equations:

Horrat =CVobtCVtg,

CVtg = 2(1−0.5×log𝐶)

,

(1)

where CVobt is the coefficient of variation of intralaboratoryreproducibility from validation data, CVtg is the targetcoefficient of variation, and 𝐶 is the mass fraction expressedas exponent of 10 [14].

The overall mean concentrations obtained in the repro-ducibility study were used to calculate recovery expressed aspercent.

Additionally, depending on the availability of the samples,the test on the repeatability and within-laboratory repro-ducibility was performed on target commercial samples.

3. Results and Discussion

3.1. Method Optimisation. The method presented in thispaper is based on the derivatisation approach from ISOnorm [11], which proved to be fit for purpose. Still, as thescope of this method is wider in terms of the numberof analytes included, the parameters of the detection andseparation had to be reoptimised. During the adaptation ofdetection conditions, two derivatisation agents used com-monly in the detection of ionophoreswere compared: vanillinand DMAB. As expected from bibliographical data [10],DMAB-derivatives gave higher signals. This phenomenonwas observed for all ionophores but was most pronouncedfor the analytes giving high response anyway (especiallyMON). Therefore, it was difficult to find such conditions ofsimultaneous detection of all target compounds that MADand SMD gave acceptable signals and MON did not saturatethe detector at the same time. For this reason, vanillin waschosen as the derivatisation reagent, after confirming that itgives acceptable signals for all ionophores.

The next step was the optimization of derivatisationconditions (concentrations and flow rates of reagents, tem-perature of reaction). Again, the aim of these experimentswas to find the conditions optimal for the coccidiostats mostdifficult to detect. As MAD and SMD are not that easilyderivatised as the ionophores included in the ISO method

(MON, NAR, and SAL) [11], it was necessary to heat thereaction coil up to 110∘C. To prolong the stability of thereagents and avoid the necessity of cooling them, it wasdecided to prepare both solutions (vanillin and sulfuric acid)in separate bottles.

In the optimization of chromatographic conditions, it wasvery important to obtain complete separation of semduram-icin and monensin. As presented in Figure 2, SMD is elutingbetween two forms of MON. Potentially, even low levelsof monensin could interfere with quantification of SMD,if not separated sufficiently. The acceptable separation wasobtained with column Luna C18(2) and very flat gradient ofmethanol/buffer (details are in Figure 2). With the fused corecolumn (Kinetex C18) shorter analysis time with isocraticelution was obtained. Although it was observed that theresolution was slightly worse in case of Kinetex column,this compromise in chromatographic performance was notsignificant. Taking into account sample throughput andreagent consumption, this column was used in all the nextexperiments.

The sensitive detection of vanillin derivatives enables thedirect analyses of sample extracts without any purification.In comparison to the ISO norm, only slight modificationsof the protocol were implemented. The sample weight andthe volume of methanol for extraction were decreased,which reduced the cost of analysis and its impact on theenvironment. The change of solvent for injection (methanolinto DMSO) improved peak shapes and stability of analytesin the extract.

As presented in Figure 3, no interferences were observedduring analyses of blank samples. The good performance ofthe method, as well as its labour efficiency, is a consequenceof the sensitive and selective detection system.

3.2. Validation and Verification of the Method. During thestatistical evaluation of obtained results, it was shown that theprecision of the determination of methyl-monensin is muchlower than that of other analytes. In consequence, its use asinternal standard was not beneficial in terms of quantifica-tion.Therefore, it was decided to calculate the results directlyfrom ionophores’ peak areas and use methyl monensin onlyfor qualitative control of the analytical process.

The results of validation study are presented in Tables2 and 3. The linearity range covers concentrations includedin authorization documents (Table 1). The analysis of blanksamples proved the selectivity of the method and its accept-able sensitivity (Figure 3).

The obtained values of limit of quantification (1.0–5.0mg/kg) are slightly higher than the ones from normalizedmethods [10–13]. A lower limit of quantification is possible toachieve; it was not, however, included in validation study.

The recovery and precision evaluation was performedusing the standard scheme, applied also in the residue control[15, 16]. The criterion for the acceptability of the method wastheHorrat value, which should not exceed 1 in reproducibilityconditions. In the case of this study, where only inhousevalidation was performed, it was expected that the variationwould be closer to repeatability target value (around 0.66).


Table 1: The authorization of ionophore coccidiostas in European Union (as for 09/04/2013) [1].

Coccidiostat Species or category of animalContent range, mg ofactive substance/kg ofcomplete feedingstuff

Lasalocid

Turkeys (<12 weeks) 90–125Chickens for fattening 75–125

Chickens reared for laying (<16 weeks) 75–125Pheasants, guinea fowl, quails and partridges except laying birds thereof. 75–125

Maduramicin Chickens for fattening 5-6Turkeys (<16 weeks) 5-5

MonensinChickens for fattening 100–125

Chickens reared for laying (<16 weeks) 100–125Turkeys (<16 weeks) 60–100

Narasin Chickens for fattening 60–70

Salinomycin Chickens for fattening 50–70Chickens reared for laying (<12 weeks) 50–50

Semduramicin Chickens for fattening 20–25

30242220181614121086420−10−5

05

101520253035404550556065

RT (min)

(𝜇V

)

Lasa

loci

d

Mon

ensin

Mad

uram

icin

Salin

omyc

inN

aras

in

×103

Sem

dura

mic

in

2826

(a)

302826242220181614121086420RT (min)

−10−5

05

1015202530354045505560706575

(𝜇V

)

×103

Lasa

loci

d

Sem

dura

mic

in Mon

ensin

Mad

uram

icin

Salin

omyc

inN

aras

in

(b)

Figure 2: The comparison of the separation of six ionophore coccidiostats with traditional porous column and fused core technology. (a)Chromatogram obtained on Phenomenex Luna C18(2) column (150 × 4.6mm, 3𝜇m) with gradient elution of methanol (A) and 0.02MKH2

PO4

(B) 0–10min: 88% A, 14–19min: 90% A, 22–32min: 88% A; flow rate 0.7mL/min. (b) Chromatogram obtained on PhenomenexKinetex C18 column (150 × 4.6mm, 2.6 𝜇m) with isocratic elution with methanol and 0.02M KH

2

PO4

(88 : 12, v : v); flow rate 0.7mL/min.


Table 2: The results of in house validation: sensitivity and linearity data.

LOD, mg/kg LOQ, mg/kg Range, mg/kg Calibration curve 𝑅

LAS 0.47 5.0 5–200 𝑦 = 1700𝑥 − 254 0.999MAD 0.65 1.0 1–40 𝑦 = 376𝑥 − 11.3 0.999MON 0.34 5.0 5–200 𝑦 = 1996𝑥 − 33.4 0.999NAR 0.23 5.0 5–200 𝑦 = 568𝑥 − 157 0.999SAL 0.25 5.0 5–200 𝑦 = 5121𝑥 − 61.2 0.999SMD 0.42 5.0 5–200 𝑦 = 208𝑥 − 3.484 0.999

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20

100

2030405060708090

−10

RT (min)

(𝜇V

)

×103

Met

hyl-m

onen

sin (I

S)

(a)

10 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20

100

2030405060708090

−10

RT (min)

(𝜇V

)

×103

Met

hyl-m

onen

sin (I

S)

Lasa

loci

d

Sem

dura

mic

in

Mon

ensin

Mad

uram

icin

Salin

omyc

inN

aras

in

(b)

Figure 3: Chromatograms of blank poultry feed sample (a) and feed sample spikedwith 50mg/kg LAS,MON,NAR, SAL, SMD, and 10mg/kgMAD (b).

As it may be seen from Table 2, the highest obtained Horratis 0.73.

In the case of the analyses of commercial target samples,the precision of the protocol is slightly lower. As the sampletreatment is really easy and straightforward, the extractionefficiency seems to be the key factor influencing the methodperformance. Since also these commercial samples results areacceptable, they prove the fitness for purpose of the presentedprotocol.

Also the previously publishedmethods using postcolumnderivatisation approach [11–13] give reliable results and arefast and easy to perform. In contrast, the application ofother derivatisation protocolsmakes it impossible to omit thecleanup with solid phase extraction and often significantly

impairs both qualitative (limit of detection) and quantitative(precision) performance of the methods [3, 17].

The developed method was successfully verified exter-nally, by the proficiency tests organized by Ducares (TheNetherlands). National Veterinary Research Institute hasparticipated in two rounds of the programme concerningthe determination of monensin and salinomycin in feeds andobtained z-scores from −1.0 to 1.9.

4. Conclusions

The authors present an effective method for the deter-mination of polyether ionophores in the feed. Thanks to


Table 3: The results of in house validation: recovery and precision of the determination of six ionophore coccidiostats in feed samples.

CoccidiostatTarget

concentration,mg/kg

Meanconcentration,

mg/kgRecovery, % RSDr, % RSDR, % Horrat∗

LAS

5.02550100CFS

5.4224.344.485.860.9

109978986—

6.52.00.41.71.7

—4.02.83.45.9

0.500.400.320.430.68

MAD

1.05.01020

1.104.8310.519.2

1109710596

5.22.51.71.6

—5.15.43.4

0.330.410.470.33

MON

5.02550100

5.0023.249.794.3

100939994

2.82.01.31.0

—4.45.32.7

0.180.440.590.34

NAR

5.02550100CFS

4.6023.347.791.247.2

92939591—

5.42.40.44.03.8

—4.61.34.16.5

0.420.470.140.510.73

SAL

5.02550100CFS

4.5223.950.591.163.3

909510191—

3.22.31.54.83.6

—4.35.25.15.2

0.250.440.580.640.61

SMD

5.02550100

4.6521.348.589.6

93859790

6.61.32.01.1

—5.83.63.2

0.510.580.400.40

RSDr: relative standard deviation of repeatability, RSDR: relative standard deviation of in house reproducibility.CFS: commercial target feed sample.∗Horrat ratio is calculated from repeatability for the lowest spiking level and from in house reproducibility for all other samples.

the modification of chromatographic separation (use of core-shell column and mobile phase at pH 7.0) and derivatisationstep (increased temperature of the reactor) in comparisonto ISO norm, the developed method allows simultaneousdetermination of six polyether antibiotics. The results ofvalidation and proficiency test confirm the applicability of themethod in the routine feed analysis.

Conflict of Interests

The authors declare that there is no conflict of interestsregarding the publication of this paper.

References

[1] “European Union Register of Feed Additives pursuant to Regu-lation (EC) No 1831/2003,” Edition 157, 2013.

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[4] M. R. Coleman, J. W. Moran, and D. H. Mowrey, “Liquid chro-matographic determination of monensin in premix and animalfeeds: collaborative study,” Journal of AOAC International, vol.80, no. 4, pp. 693–702, 1997.

[5] U. Vincent, F. Serano, M. J. G. de la Huebra, and C. von Holst,“Determination of semduramicin in poultry feed additive,premixture and compound feed by liquid chromatography andUV spectrophotometric detection after post-column derivati-sation,” Journal of Pharmaceutical and Biomedical Analysis, vol.61, pp. 150–155, 2012.

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[9] U. Vincent, M. Chedin, S. Yasar, and C. von Holst, “Deter-mination of ionophore coccidiostats in feedingstuffs by liquidchromatography-tandem mass spectrometry. Part I. Applica-tion to targeted feed,” Journal of Pharmaceutical and BiomedicalAnalysis, vol. 47, no. 4-5, pp. 750–757, 2008.

[10] H. Campbell, G. Nayeri, J. M. Costa et al., “Determinationof monensin, narasin, and salinomycin in mineral premixes,supplements, and animal feeds by liquid chromatography andpost-column derivatization: collaborative study,” Journal ofAOAC International, vol. 89, no. 5, pp. 1229–1242, 2006.

[11] Animal feeding stuffs, “Determination of monensin, narasinand salinomycin contents. Liquid chromatographic methodusing post-column derivatisation,” ISO 14183, 2005.

[12] Animal feeding stuffs, “Determination of maduramicin-ammonium by reversed-phase HPLC using post-columnderivatisation,” EN 15781, 2009.

[13] Animal feeding stuffs, “Determination of semduramicin con-tent. Liquid chromatographic method using a “tree” analyticalapproach,” EN 16158, 2012.

[14] M. Thompson, “Recent trends in inter-laboratory precisionat ppb and sub-ppb concentrations in relation to fitness forpurpose criteria in proficiency testing,” Analyst, vol. 125, no. 3,pp. 385–386, 2000.

[15] M. Thompson, S. L. R. Ellison, and R. Wood, “Harmonizedguidelines for single-laboratory validation of methods of analy-sis (IUPACTechnical Report),”Pure andApplied Chemistry, vol.74, no. 5, pp. 835–855, 2002.

[16] “Commission Decision 2002/675/EC of 12 August 2002 imple-menting Council Directive Directive 96/23/EC concerning theperformance of analytical methods and the interpretation ofresults,” Official Journal of the European Communities, vol. 221,pp. 8–36, 2002.

[17] G.Dusi andV.Gamba, “Liquid chromatographywith ultravioletdetection of lasalocid, monensin, salinomycin and narasinin poultry feeds using pre-column derivatization,” Journal ofChromatography A, vol. 835, no. 1-2, pp. 243–246, 1999.

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