DART mass spectrometry: a rapid tool for the identification of feed additives containing coccidiostats as active substances.

Feed additives require pre-market authorisation prior to their use in the EU. For the group of coccidiostats, the EU regulations authorising these products include specifications for these substances and the major components of the feed additive formulations. Feed business operators can use only feed additives that meet these criteria. The traceability of these products is supported by the allocation of specific identification numbers that need to be printed on the feed additive label along with other information. In the present study, Direct Analysis in Real Time mass spectrometry (DART-MS) was applied to investigate the correct characterisation of feed additives that contain coccidiostats as active substances. The results of the study demonstrated that this technique allows an unequivocal identification of the active substances in the feed additive formulations when combining DART with high-resolution mass spectrometry. In addition, two feed additives containing the same coccidiostat, but different excipients could be correctly classified according to their composition. The method protocol is very simple and comprises two steps, namely the extraction of the feed additives with an organic solvent and the subsequent measurement with DART-MS. For the evaluation of the MS spectra, chemometrics was applied offering an effective method for classification. Chemometric models were established with nominal masses obtained from the analysis of the samples, thus showing that these feed additives could be correctly classified even using low-resolution mass spectrometry. Moreover, we demonstrated that molecule-specific stable isotope patterns obtained with low-resolution mass spectrometry could be used as an alternative tool for the confirmation of the active substance.

Determination of ionophore coccidiostats in feeding stuffs by liquid chromatography-tandem mass spectrometry. Part II. Application to cross-contamination levels and non-targeted feed.

A fit to purpose multi-analyte method for the official control of six coccidiostats (monensin sodium, salinomycin sodium, narasin, lasalocid sodium, semduramicin sodium and maduramicin ammonium alpha) at cross-contamination concentration levels in poultry, cattle, pig and calf compound feed by liquid chromatography tandem mass spectrometry (LC-MS/MS) has been developed and in-house validated. The corresponding maximum levels have been recently introduced by European legislation. The method developed involved a simple extraction of the coccidiostats from the feed samples followed by centrifugation and filtration of the supernatants for all matrices. For calf feed an additional de-fattening step of the filtrated supernatants with n-hexane was necessary. The resulting supernatants were submitted to chromatographic analysis. The analytes were quantified by a modified approach of the standard additions technique applied to the extracts, hence allowing a workload comparable to matrix-matched standard calibration curves. A further simplification of this technique was reached by applying the same addition levels of the target analytes for different concentration ranging from 0.5× maximum level up to 2.5× maximum level (universal approach). The concentration independent intermediate precision expressed in terms of relative standard deviation varied between 3 and 12% (except for maduramicin ammonium alpha and semduramicin sodium up to 21%) and the recovery rates ranged from 80 to 111%, depending on the target analyte and matrix. The limits of detection (LOD) and limits of quantification (LOQ) were different for the various analyte/matrix/instrument combinations but all LOQs were in the 0.01-0.65 mg kg(-1) range, hence well below the target concentrations of each analyte. Based on the obtained method performance characteristics the method is considered fit for the intended purpose.

Determination of ionophore coccidiostats in feedingstuffs by liquid chromatography-tandem mass spectrometry Part I. Application to targeted feed.

A new and fit to purpose multi-analyte method for the determination of six coccidiostats (monensin A, salinomycin, narasin composed of its principal component narasin A and its minor component narasin I, lasalocid, semduramicin and maduramicin) in poultry and cattle compound feed by liquid chromatography tandem mass spectrometry (LC-MS/MS) has been developed and in-house validated. The concentration level of the target analytes at which the validation experiments have been carried out varied between 1 and 9 mg kg(-1). The method developed involved a simple extraction of the coccidiostats from the feed samples followed by a clean-up by solid-phase extraction prior to chromatographic analysis. The analytes were quantified either by matrix-matched standards or by the standard addition technique, obtaining the following performance profile of the method for the various analyte/matrix combinations. When quantifying against matrix-matched standards, the concentration independent intermediate precision expressed in terms of relative percentage standard deviation varied between 4 and 10% and the relative percentage recovery rates ranged from 86 to 120%, depending on the target analyte and matrix. When using the standard addition technique, the corresponding values for the intermediate precision varied between 2 and 8% and the relative percentage recovery rate ranged from 73 to 115%. The limit of detection (LOD) and limit of quantification (LOQ) were different for the various analyte/matrix combinations but were in all cases below 0.014 and 0.046 mg kg(-1), respectively. Based on the obtained method performance characteristics, the method is considered suitable for the determination of ionophore coccidiostats in target feed. The main field of application of the validated method is to enforce European legislation regarding the authorisation of coccidiostats, focusing on the measurement at the authorised levels and at low level in feed during the withdrawal period at which the coccidiostats must not be added to the feed. Overall, the method proposed appears to be appropriate as a confirmatory method for the monitoring of these six ionophore coccidiostats and can therefore be considered as complementary to the official HPLC-UV methods.