Determination of Eight Coccidiostats in Eggs by Liquid-Liquid Extraction-Solid-Phase Extraction and Liquid Chromatography-Tandem Mass Spectrometry.

A method for the simultaneous determination of robenidine, halofuginone, lasalocid, monensin, nigericin, salinomycin, narasin, and maduramicin residues in eggs by liquid chromatography-tandem mass spectrometry (LC-MS/MS) was developed. The sample preparation method used a combination of liquid-liquid extraction (LLE) and solid-phase extraction (SPE) technology to extract and purify these target compounds from eggs. The target compounds were separated by gradient elution using high-performance liquid chromatography (HPLC) and ultra-performance liquid chromatography (UPLC). Tandem mass spectrometry was used to quantitatively and qualitatively analyze the target compounds via electrospray ionization (ESI+) and multiple reaction monitoring mode. The HPLC-MS/MS and UPLC-MS/MS methods were validated according to the requirements defined by the European Union and the Food and Drug Administration. The limits of detection and limits of quantification of the eight coccidiostats in eggs were 0.23-0.52 µg/kg and 0.82-1.73 µg/kg for HPLC-MS/MS, and 0.16-0.42 µg/kg and 0.81-1.25 µg/kg for UPLC-MS/MS, respectively. The eggs were spiked with four concentrations of the eight coccidiostats, and the HPLC-MS/MS and UPLC-MS/MS average recoveries were all higher than 71.69% and 72.26%, respectively. Compared with the HPLC-MS/MS method, utilizing UPLC-MS/MS had the advantages of low reagent consumption, a short detection time, and high recovery and precision. Finally, the HPLC-MS/MS and UPLC-MS/MS methods were successfully applied to detect eight coccidiostats in 40 eggs.

Multiple response optimization of a QuEChERS extraction and HPLC analysis of diclazuril, nicarbazin and lasalocid in chicken liver.

A method for the simultaneous determination of three commonly used coccidiostats in chicken liver was developed, comprising a multi-residue QuEChERS (quick, easy, cheap, effective, rugged and safe) extraction step, and a liquid chromatography-ultra violet-fluorescence (HPLC-UV/FL) analysis. The QuEChERS extraction was optimized using an experimental design approach that includes a screening step to obtain the critical variables, an optimization step using multiple response surface analysis and the calculation of a desirability parameter. The optimized method was validated with fortified samples, reaching an average recovery of 91% and an overall precision of 5.5% (mean of three analytes at three levels). Limits of detection calculated on fortified samples were 20 µg kg-1 for lasalocid, 15 µg kg-1 for nicarbazin and 120 µg kg-1 for diclazuril. These values resulted at least one order of magnitude lower than the maximum allowed residue limit (MRL) of the studied coccidiostats for chicken liver.

Structural characterization of polyketides using high mass accuracy tandem mass spectrometry.

The tandem mass spectrometry techniques electron-induced dissociation (EID) and collision-activated dissociation (CAD) have been compared as tools for providing detailed structural information of polyketides. Polyketides are an important class of natural products that account for a significant proportion of the drugs currently in clinical use. Three polyketide natural products, namely erythromycin A, lasalocid A, and iso-lasalocid A, were subjected to both CAD and EID, and their fragment ions were assigned with sub-part-per-million accuracy. The number of fragment ions detected through EID was much greater than for CAD, leading to a greater amount of structural information obtained for each polyketide, albeit with a decreased signal-to-noise ratio. The effect of different bound cations on the fragment pattern of the isomers lasalocid A and iso-lasalocid A was studied, with CAD and EID performed on the [M + H](+), [M + Na](+), [M + Li](+), and [M + NH(4)](+) precursor ions. The lithiated species were found to produce the greatest degree of fragmentation and enabled detailed structural information on the isomers to be obtained. Multistage mass spectrometry (MS(3)) experiments, combining CAD and EID, could also be performed on the lithiated species, generating new fragment information which enables the two isomers to be distinguished. Combining CAD and EID for the structural characterization of polyketides will therefore be a useful tool for identifying and characterizing unknown polyketides and their biosynthetic intermediates.

Biosynthesis of lasalocid A: biochemical mechanism for assembly of the carbon framework.

Labeling experiments on the biosynthesis of the polyether antibiotic lasalocid A (1) using carboxylic acid precursors bearing 13C, 2H, and 3H labels at various positions established the following: (1) 2H or 3H at C-2 of propionate or 2H at C-2 of butyrate was partially retained at C-12 and C-14 of 1, respectively. (2) 2H at C-2 of propionate or at C-2 and C-3 of succinate did not label C-10. These and earlier data [Hutchinson, C. R., Sherman, M. M., Vederas, J. C., & Nakashima, T. T. (1981) J. Am. Chem. Soc. 103, 5953; Hutchinson, C. R., Sherman, M. M., McInnes, A. G., Walter, J. A., & Vederas, J. C. (1981) J. Am. Chem. Soc. 103, 5956] are consistent with a hypothesis for the stereochemical control of lasalocid A biosynthesis, whose main tenets are that the configuration of C-12 and C-14 is determined by the stereoselectivity of the carbon chain forming condensation between acyl thio ester and 2-carboxyacyl thio ester intermediates and that the configuration of C-11 and C-15 results from the reduction of 2-keto thio ester intermediates with opposing stereospecificities.

Collaborative study of a modified extraction procedure for lasalocid sodium in feeds by microbiological assay.

A collaborative study was conducted on a modified extraction technique in the microbiological determination of lasalocid sodium in finished poultry feeds. The feed is warmed briefly with pH 4.7 buffer and the drug is extracted with ethyl acetate. The extract is washed with HCl and NaOH and evaporated to dryness, and the residue is dissolved in hexane, partitioned into methanol-water (3+1), and diluted to assay range at a final alcohol concentration of 25% (v/v). Seven laboratories participated in the study. Average recoveries of lasalocid sodium added at 0.01035, 0.00863, and 0.0069% ranged from 93 to 99% for mash feeds and from 86 to 95% for pellets. The coefficients of variation averaged 11.2% for mashes and 9.8% for pellets. The modified extraction technique has been adopted as official first action.