LC/MS confirmation of ionophores in animal feeds.

A liquid chromatographic/mass spectrometric (LC/MS) electrospray confirmation method has been developed to confirm 4 ionophores (monensin, lasalocid, salinomycin, and narasin) in a variety of animal feeds using a single quadrupole mass spectrometer. The sodium ions of these compounds are dominant in the electrospray mass spectrum. Using optimized "in-source" collision induced dissociation, characteristic fragment ions seen previously using MS/MS can be observed. The drugs were extracted from the feed matrix using hexane-ethyl acetate and isolated using a silica solid-phase extraction cartridge. These ionophores were confirmed in both medicated feeds and nonmedicated feeds fortified with these drugs at the 1-50 ppm level. In addition, this method was used to confirm residues of monensin in a nonmedicated feed that was collected from a feed mill immediately after the production of a similar feed that was medicated with high levels of monensin.     

Rapid method for the simultaneous determination of six ionophores in feed by liquid chromatography/mass spectrometry

A simple and highly sensitive LC/MS method was developed for the simultaneous determination of six ionophores--lasalocid, monensin, laidlomycin, maduramycin, salinomycin, and narasin--in feed. The procedure involved extraction of 1 g of feed with 4 mL of methanol-water (9 + 1, v/v) by shaking on a platform shaker for 45 min. After centrifugation, the extracts were diluted with methanol-water (75 + 25, v/v) and analyzed without any cleanup. The analysis was performed on a Betasil C18 column (150 x 4.6 mm id, 5 pm particle size) connected to an LC/MS system operated in the atmospheric pressure chemical ionization (APCI) mode. We believe this to be the first method that uses the APCI mode for the analysis of ionophores. The mobile phase consisted of 50 mM ammonium acetate as solvent A and acetonitrile-methanol (7 + 3, v/v) as solvent B in a gradient run. Excellent recoveries of 81-120% were found for all compounds at fortification levels of 1-200 microg/g, with RSD < or =15% (except 17% for maduramycin at 2 and 5 microg/g, and 16% for salinomycin at 1 microg/g). At 0.5 microg/g, recoveries of 87-119% were obtained, with RSD < or =20%. However, recovery of lasalocid was 133% and salinomycin 79% in sow and horse feed, respectively. Average RSD values of lasalocid and salinomycin were 22 and 21%, respectively. Finally, proficiency test samples analyzed with the method demonstrated favorable agreement with the certified values.     

A new approach to the analysis of nicarbazin and ionophores in eggs by HPLC/MS/MS

An HPLC/MS/MS method has been developed and validated for the quantification and confirmation of nicarbazin and ionophores (lasalocid, monensin, salinomycin, and narasin) in eggs. Nicarbazin is determined in the negative electrospray mode with a basic mobile phase that supports creation of negative ions. Consequently, our ability to maintain instrument sensitivity over time has significantly improved. The analysis of the ionophores is done in the positive electrospray mode using ammonium buffer for HPLC separation. Monitoring ammonium adduct parent ions resulted in enhanced sensitivity and better reproducibility of the ionophore analysis. The validation of this improved HPLC/MS/MS method for the detection of nicarbazin and the ionophores demonstrated excellent precision of below 10% RSD and lower LOD values (microg/kg) for nicarbazin (0.018), lasalocid (0.015), monensin (0.015), salinomycin (0.033), and narasin (0.039).     

Multi-residue confirmatory method for the determination of twelve coccidiostats in chicken liver using liquid chromatography tandem mass spectrometry

The confirmatory LC-MS/MS method for the determination of residues for twelve coccidiostats including ionophore antibiotics (lasalocid, maduramycin, monensin, narasin, salinomycin, semduramycin) and chemical coccidiostats (clazuril, decoquinate, diclazuril, halofuginone, nicarbazin and robenidine) in poultry liver has been developed. The sample preparation was based on extraction with acetonitrile, defatting with Alumina columns and clean-up on Oasis HLB spe. The separation of analytes was performed on PhenylHexyl column with a gradient of acetonitrile, methanol and the ammonium formate pH 4.0. For all analytes, at least 2 diagnostic fragmentation ions were monitored. The validation, performed according to the CD 2002/657/EC, proved the suitability of the method for the confirmatory analysis of the coccidiostats. For lasalocid, however, low reproducibility was observed and the proper quantification could not be performed with this method.     

Validation of a multi-residue liquid chromatography–tandem mass spectrometry confirmatory method for 10 anticoccidials in eggs according to Commission Decision 2002/657/EC

A liquid chromatography–tandem mass spectrometric (LC–MS/MS) method for the simultaneous detection and confirmation of halofuginone, robenidine, diclazuril, nicarbazin, monensin, narasin, lasalocid, salinomycin, maduramicin and semduramicin in whole egg has been developed and validated. The anticoccidial residues were extracted by acetonitrile, evaporated and dissolved in a sodium acetate/acetonitrile mixture. Then, the samples were injected on a C8 column in a gradient mode. Diclazuril-bis, DNC-d8 and nigericin were used as internal standards. The results of the full validation in accordance with the guidelines of the Commission Decision no 2002/657/EC are presented. This rapid and sensitive method was found suitable to confirm the anticoccidials at 1 and at 75 μg kg⁻¹ for the MRL compound lasalocid.     

Rapid forensic selected reaction monitoring liquid chromatography/mass spectrometry determination of ionophore antibiotics found at toxic levels in animal feeds

A rapid, accurate, and selective method was developed for the forensic determination of ionophore antibiotics in animal feeds. A simple extraction procedure and liquid chromatography/tandem mass spectrometry (LC/MS/MS) in the selected reaction monitoring (SRM) mode were used for rapid identification and confirmation of monensin and lasalocid in feed samples and for quantitation of monensin. Extracts from a homogenous portion of ground feeds were prepared using liquid-solid extraction and liquid-liquid extraction techniques. Feed extracts were further purified by a simple defatting and solvent wash step and then concentrated to dryness. Feed extract residues were reconstituted in 1 mL LC mobile phase and a 2 microL aliquot injected into the SRM LC/MS system. The latter system used a C18, 100 x 2.0 mm, LC column coupled to a PE-Sciex API 2000 tandem triple quadrupole mass spectrometer equipped with a TurbolonSpray LC/MS interface. Feed samples were extracted and analyzed for the determination of monensin and lasalocid within a couple of hours. Control feed samples fortified with monensin at concentrations from 50 ppb to 5 ppm provided a linear response and calibration curve across this range with a correlation coefficient of 0.996.     

The determination of 5 anticoccidial drugs (nicarbazin,lasalocid, monensin,salinomycin and narasin) in animal livers and eggs by liquid chromatography linked with tandem mass spectrometry (LC-MS-MS)

A method has been developed for the simultaneous extraction and determination of five of the most commonly used anticoccidial drugs (nicarbazin, lasalocid, monensin, narasin and salinomycin) from livers and eggs by LC-MS-MS. Results show good repeatability, with mean spiked recoveries for nicarbazin, lasalocid, monensin, narasin and salinomycin in poultry livers in the average range of 92-118%, and 86-110% in eggs. The detection limit is at 1 ng ml(-1) for all the named compounds and a quantitation level of 2.5 ng g(-1) has been achieved. A high throughput of samples is achievable using this method which allows the analysis of up to 40 samples by one analyst in a day.     

Determination of narasin and monensin in bovine, swine, and chicken tissues by liquid chromatography with tandem mass spectrometry: first action 2011.24

The single-laboratory validation (SLV) of an LC-MS/MS method for determination and confirmation of two ionophores, narasin and monensin, in animal tissues is described. The data demonstrated linearity of matrix-matched calibration curves using a weighted (1/x) regression and selectivity of the method for narasin and monensin in the presence of lasalocid, salinomycin, maduramycin, nicarbazin, and sulfadiazine. Recoveries varied from 86.2 to 103.5% for narasin and 89.1 to 105.1% for monensin. Intertrial repeatability precision [relative standard deviation of repeatability (RSDr)] varied from 3.9 to 13.8% for narasin and 3.3 to 16.3% for monensin in fortified tissue. Precision of the method was verified in incurred tissues. The LOQ of the method was validated and ranged from 0.45 ng/g in milk, to 4.0 ng/g in chicken fat, but was 0.75 ng/g for most tissues. Two confirmatory ions for each analyte were examined across all matrixes, resulting in estimated false-negative rates of 0.00% (95% confidence interval of 0.00-0.68%) for monensin ions (540 samples) compared to the U.S. and European Union (EU) acceptance criteria. The confirmatory ions for narasin demonstrated 0.00% false-negative rates (95% confidence interval of 0.00-0.58%) when compared to either the U.S. or EU criteria in 630 samples. The method was robust when small changes in method parameters were made and stability of fortified tissues, extracts, and calibration solutions were estimated. The data satisfy the requirements of the AOAC Stakeholder Panel on Veterinary Drug Residue for SLV studies, and the method was adopted Official Methods of Analysis First Action 2011.24 by the AOAC Expert Review Panel on Veterinary Drug Residues.     

The characterisation of polyether ionophore veterinary drugs by HPLC-electrospray MS.

We undertake the determination of a wide range of veterinary drug residues in a range of animal products. Various screening analyses are employed, followed by HPLC-API (atmospheric pressure ionisation)-MS for the unequivocal confirmation of significant positives. EU legislation for the use of GC-MS as a confirmatory technique requires the successful monitoring of at least four diagnostic ions and although no such requirement exists for HPLC-MS confirmation, a similar requirement would seem appropriate. Until recently, reports describing the electrospray MS confirmation of residues of the polyether ionophores have been based on monitoring one or two ions. We have found that the addition of ammonium acetate to the HPLC mobile phase, in conjunction with 'cone voltage' or 'skimmer' assisted fragmentation, is a convenient way of producing additional diagnostic ions from polyether ionophore compounds, without compromising the overall sensitivity. Results for lasalocid, the most widely used compound, are presented. Electrospray MS data and acquisition parameters for lasalocid, monensin, narasin and salinomycin are described. The advantage of this analytical approach is that it may be used to generate confirmatory data using a single quadrupole MS system, without the need for advanced MS instrumentation, e.g., MS-MS.     

Development and validation of an SPE methodology combined with LC-MS/MS for the determination of four ionophores in aqueous environmental matrices

A multi-residue analytical methodology has been established for the determination of the four ionophores: lasalocid, monensin, salinomycin and narasin in aqueous environmental matrices, using nigericin as internal standard. The samples were filtrated prior to solid phase extraction. All compounds were measured using liquid chromatography coupled to tandem mass spectrometry applying electro spray ionisation. The absolute recoveries ranged from 92 to 110% (relative standard deviation: 2–14%) for spiked river water. The final method allowed for detection of ionophores down to a few ng/L in natural water bodies with LOQs for the entire methodology being 40, 49, 67, and 14 ng/L for lasalocid, monensin, salinomycin, and narasin, respectively.     

Liquid chromatography with ultraviolet detection of lasalocid, monensin, salinomycin and narasin in poultry feeds using pre-column derivatization

A rapid and very effective analytical procedure for the simultaneous determination of four polyether antibiotics (PEs) lasalocid, monensin, salinomycin and narasin in poultry feeds was tested. PEs were extracted from samples using methanol and without and clean-up derivatized with 2,4-dinitrophenylhydrazine (DNP) in an acidic medium at 55 degrees C. The derivatization mixture was analyzed directly on an ODS column (150 x 4.6 mm, 5 microns) with methanol--1.5% aqueous acetic acid (90:10, v/v) as eluent and UV detection was carried out at 305/392 nm. The recoveries of the PEs from spiked samples were 85-100% with RSDs of 4-10% in a concentration range of 50-150 mg/kg.     

Simultaneous determination of polyether ionophores, macrolides and lincosamides in hen eggs by liquid chromatography-electrospray ionization tandem mass spectrometry using a simple solvent extraction

A liquid chromatography-electrospray ionization tandem mass spectrometric (LC-ESI-MS/MS) method was developed and validated for the determination of residues of 6 polyether ionophores (lasalocid, maduramicin, monensin, narasin, salinomycin, semduramicin), 3 macrolides (erythromycin, tylosin, clarithromycin) and 1 lincosamide (lincomycin) in eggs. Nigericin was used as qualitative internal standard. Samples were deproteinizated/extracted with acetonitrile without pH adjustments. Aliquots of the extracts were evaporated and reconstituted for injection in the instrument operated in positive multiple reaction monitoring (MRM) mode. The stability of the antibiotics and the intensity of the formed ions were considered in order to select a suitable solvent for the reconstitution of the obtained dry extracts. No clean-up steps were required and matrix effects were controlled by sample dilution, selection of appropriate chromatographic conditions and reduced injection volume. Good within-laboratory reproducibility was obtained, with relative standard deviations (RSD(R)) from 4.0 (semduramicin at 5 μgkg(-1)) to 18.6 (erythromycin at 25 μgkg(-1)) for the ionophores and macrolides. Lincomycin showed the least precise results, with a maximum RSD(R) of 20.2% at 75 μgkg(-1)). Satisfactory decision limits (CCα) and detection capabilities (CCβ) were also attained. Method limits of detection (LODs) from 0.04 (salinomycin) to 1.6 μgkg(-1) (lincomycin) were achieved. Method limits of quantification (LOQs) were from 0.14 to 5.3 μgkg(-1) for the same drugs, respectively. All the LOQs, except that obtained for maduramicin were remarkably below the lowest validation level. The proposed method is suitable for routine application in commercial egg samples.     

Fast liquid chromatography/multiple‐stage mass spectrometry of coccidiostats

Drugs that are used as medicines and also as growth promoters in veterinary care are considered as emerging environmental contaminants and in recent years concern about their potential risk to ecosystems and human health has risen. In this paper we used a method based on liquid chromatography/electrospray tandem mass spectrometry to analyze eight coccidiostatic compounds: diclazuril, dinitrocarbanilide (the main metabolite of nicarbazin), robenidine, lasalocid, monensin, salinomycin, maduramicin and nasarin. Multiple‐stage mass spectrometry (MSⁿ) based on the precursor ions [M+Na]⁺ (polyether ionophores), [M+H]⁺ (robenidine) and [M–H]^? (diclazuril and dinitrocarbanilide) was used to study the fragmentation of these compounds. MSⁿ data and genealogical relationships were used to propose a tentative assignment of the different fragment ions. Loss of water, decarboxylations, ketone β‐cleavages and rearrangement of cyclic ethers and amide groups were some of the fragmentations observed for these compounds. Liquid chromatography with a sub‐2 µm particle size column was coupled to tandem mass spectrometry (LC/MS/MS) allowing the separation of these compounds in less than 7 min. Method detection limits ranging from 11 to 71 ng L^?1 and run‐to‐run values in terms of relative standard deviation (RSD) (up to 12%) were obtained. Copyright © 2009 John Wiley & Sons, Ltd.     

Periodicity in collision-induced and remote-bond activation of alkali metal ions attached to polyether ionophores

The collision-induced dissociation of lasalocid and monensin A bound to alkali metal cations has been investigated using electrospray MS/MS. The binding affinity for the metal cations, as measured with collision-induced dissociation, was found to depend on their ionic radius, decreasing with increasing radius. Monensin A was observed to have a greater binding affinity for alkali metal cations than lasalocid acid.     

Fragmentation study of salinomycin and monensin A antibiotics using electrospray quadrupole time-of-flight mass spectrometry

The fragmentation pathways of two selected ionophore antibiotics, salinomycin and monensin A, were studied using electrospray (ES) orthogonal acceleration quadrupole time-of-flight mass spectrometry in positive-ion mode. The identity of fragment ions was determined by accurate-mass measurements. In ES mass spectra, ion signals of relatively high intensity were observed for [M+Na](+) and [M-H+2Na](+) for each antibiotic. Each of the ion species [M+Na](+) and [M-H+2Na](+) for salinomycin and [M-H+2Na](+) for monensin A were isolated in turn and subjected to fragmentation. In the fragmentation of [M+Na](+) and [M-H+2Na](+) from salinomycin, only Cbond;C single bond cleavage and dehydration were observed. Product ion mass spectra obtained from [M-H+2Na](+) of monensin A showed that ether ring opening, Cbond;C single bond cleavage and dehydration fragmentations had occurred. Fragment ions containing two sodium atoms were observed in the product ion mass spectrum of [M-H+2Na](+) from salinomycin, but not from monensin A. Both type A (containing the terminal carboxyl group) and type F (containing the terminal hydroxyl group) fragment ions were observed in the product ion mass spectra of sodium adduct ions of salinomycin and monensin A.     

Single-laboratory validation of a multiplex flow cytometric immunoassay for the simultaneous detection of coccidiostats in eggs and feed

Coccidiostats are authorized in the European Union (EU) to be used as poultry feed additives. Maximum (residue) levels (M(R)Ls) have been set within the EU for consumer and animal protection against unintended carry-over, and monitoring is compulsory. This paper describes the single-laboratory validation of a previously developed multiplex flow cytometric immunoassay (FCIA) as screening method for coccidiostats in eggs and feed and provides and compares different approaches for the calculation of the cut-off levels which are not described in detail within Commission Decision 2002/657/EC. Comparable results were obtained between the statistical (reference) approach and the rapid approaches. With the most rapid approach, the cut-off levels for narasin/salinomycin, lasalocid, diclazuril, nicarbazin (DNC) and monensin in egg, calculated as percentages of inhibition (%B/B0), were 60, 32, 76, 80 and 84, respectively. In feed, the cut-off levels for narasin/salinomycin, lasalocid, nicarbazin (DNC) and monensin were 70, 64, 72 and 78, respectively, and could not be determined for diclazuril. For all analytes, except for diclazuril in feed, the rate of false positives (false non-compliant) in blank samples was lower than 1 %, and the rate of false negatives (false compliant) at the M(R)Ls was below 5 %. Additionally, very good correlations (r ranging from 0.994 to 0.9994) were observed between two different analysers, a sophisticated flow cytometer (FlexMAP 3D^®) and a more cost-efficient and transportable planar imaging detector (MAGPIX^®), hence demonstrating adequate transferability.     

Determination of Coccidiostats in Milk Products by LC–MS and Its Application to a Fermentation Experiment

A liquid chromatographic tandem mass spectrometric (LC–MS–MS) method for the determination of five chemical coccidiostats (decoquinate, diclazuril, halofuginone, nicarbazin, and robenidine) and five ionophore coccidiostats (maduramicin, monensin, narasin, salinomycin, and semduramicin) in yoghurt, kefir, and sour cream is presented. Lasalocid, the sixth ionophore listed in 124/2009/EC was not included because of its extremely dissimilar behavior during sample preparation. Main steps of the method include extraction with acetonitrile, centrifugation, clean-up on Oasis HLB solid phase extraction cartridge, evaporation under nitrogen stream, and LC–MS–MS determination. Selectivity, linearity, sensitivity, accuracy, repeatability, within-laboratory reproducibility, limit of determination, and limit of quantitation were determined during the validation procedure. The method proved to be applicable for both qualitative and quantitative determination of the ten above-mentioned target compounds. In our in-house fermentation experiments, milk fortified with coccidiostats was fermented to get yoghurt, kefir, and sour cream. Our results show that the coccidiostat content did not change significantly during fermentation for any of the target compounds.     

Fragmentation studies on lasalocid acid by accurate mass electrospray mass spectrometry

Lasalocid acid is an important polyether ionophore veterinary drug. Polyether ionophores have been the subject of MS study for many years, but this is the first rigorous study of the complex fragmentation processes occurring in ESI MS/MS for lasalocid, underpinned by high-resolution accurate-mass measurement. Initial low-resolution analyses were performed on an ion-trap instrument. High-resolution analyses were performed on a Fourier-transform ion cyclotron resonance mass spectrometer. The MS/MS analysis of the pseudo-molecular ion shows that fragment ions are produced either by beta-elimination or by neutral losses of water. Additional ions were observed in the source dissociation analysis, indicating that additional fragmentation reactions occur in the source region. Some of these ions can then undergo additional ion-ion or ion-molecule reactions before being extracted from the source. The study of both the protonated and sodiated sodium salts shows the same fragmentation pathways, with fragment ions containing two sodiums at low intensity. A fragmentation pathway of the lasalocid acid protonated sodium salt [(M-H+Na)+H]+ (m/z 613) and sodiated sodium salt [(M-H+Na)+Na]+ (m/z 635) is presented. The increased understanding afforded by this study will help in the development of unequivocal analytical methods for lasalocid and related polyether ionophore veterinary drugs.     

Simultaneous determination of five polyether ionophores using liquid chromatography with one-step fluorescent derivatization

We present a selective method for simultaneous determination of five polyether ionophores such as salinomycin (SAL), monensin (MON), narasin (NAR), semduramicin (SEM) and lasalocid (LAS) in aquatic samples using a liquid chromatography with one-step fluorescent derivatization of 2-(4-hydrazinocarbonyl-phenyl) 4,5-diphenylimidazole (HCPI) and 4-(4,5-diphenyl-1H-imidazol-2-yl) benzoyl chloride hydrochloride (DIB-Cl). Fluorescent one-step derivatization for SAL, MON, NAR and SEM using HCPI and for LAS using DIB-Cl was monitored by an LC/fluorescence detector (E(x), 340 nm; E(m), 465 nm). Chromatographic separation was performed on a TSK-GEL ODS-120T (4.6 × 150 mm, 3 μm) column using a mobile phase of 0.1% formic acid in acetonitrile and 0.5 mM ammonium formate in water (70/30, v/v). The limits of detections were 0.01 μg/mL (50 pg) for LAS, 0.05 μg/mL (250 pg) for SAL, NAR and SEM, and 0.1 μg/mL (500 pg) for MON, respectively. The recoveries for water samples were indicated to be the range of 79.6 ± 6.4 - 99.0 ± 4.4% with associated precision values (between-day for 3 days) for repeatability. Based on solid-phase extraction, the limit of quantitation values indicated 0.1 ng/mL for SAL, MON, NAR and SEM, and 0.01 ng/mL for LAS in water samples.     

Efficient and sensitive screening and confirmation of residues of selected polyether ionophore antibiotics in liver and eggs by liquid chromatography-electrospray tandem mass spectrometry.

A method using liquid chromatography tandem mass spectrometry (LC-MS-MS) with electrospray (ES) for the determination of traces of narasin, monensin and salinomycin in chicken liver and eggs was developed, validated and used for routine surveillance. The essence of this paper is to demonstrate that one single method can serve very well for two entirely different purposes, i.e., screening and confirmation. Highly reliable confirmation of the identity at low concentrations was demonstrated when residues of narasin were detected and quantified (0.2 to 11 ng g(-1)) in 50% of the Swedish eggs analysed in 1999. Four daughter ions were detected with ion ratios meeting suggested confirmation criteria for the European Union, even at 0.2 ng g(-1). The method was found to be highly cost-effective since both screening and confirmation of 98 liver samples were performed in only two analytical runs (the Swedish national surveillance scheme of 1999, report level 5 ng g(-1)). The high performance of the method for the different applications was possible due to a combination of the power of ES-LC-MS-MS, a procedure involving screening of pooled samples, and method optimisation of the work-up (automated solid phase extraction), LC and MS parameters. Validation data for narasin (0.5 to 20 ng g(-1)) in eggs are presented (accuracy 94 to 108%, relative standard deviation 4 to 10%, limit of detection 0.026 ng g(-1)). The time for an LC-MS-MS run was 4 min, corresponding to 48 s per sample in a pool.