Search

The development of a tissue residue method for an antibiotic is dependent on the history of the overall problem; the current requirements that must be met for regulatory approval; the chemistry of the compound; its physical/chemical parameters; and its microbiological spectrum for the best possible fit of all of these to ensure a procedure that will work reliably in the originator's laboratory and to the same degree of effectiveness in anyone's laboratory. A note is made of those factors that must be included as controls on the procedure and those experiments that must be included to differentiate matrix from method influences.

A liquid chromatograph was interfaced to an atomic absorption spectrometer for the detection and quantitation of maduramicin in feed matrixes at the 1-8 ppm level. Ionophores in general form strong 1:1 products with various metal cations, yielding complexes that are insoluble in water but very soluble in organic solvents. Maduramicin, a carboxylic, polyalcohol, polyether antibiotic, is labeled with the sodium cation and analyzed by atomic absorption spectroscopy (AAS). The lower limit of detection is approximately 100-200 ng maduramicin sodium salt. Feeds containing 1-8 ppm maduramicin are extracted with acetone, the extract is passed through an alumina column, the column is eluted with acetonitrile-water (90 + 10), and the eluate is analyzed for maduramicin by liquid chromatography-AAS after concentration and conversion of maduramicin to the sodium salt. Recoveries of maduramicin averaged 89.5%. Liquid chromatography with AAS detection has been shown to be a sensitive and highly specific technique for the determination of ionophores in general and maduramicin in particular.

An analytical method has been developed that can reliably measure the metabolic marker residue of lasalocid. The method monitors this marker residue in food samples to ensure that the total residue of toxicological concern is not being exceeded. Interlaboratory studies of the liquid chromatographic determinative procedure and the gas chromatographic/mass spectrometric confirmatory procedure for lasalocid sodium at the 0.7 ppm level and higher were successful.

The microbiological cylinder plate assay method for lasalocid sodium reported earlier was studied in a slightly modified form by 7 laboratories. Assay values paralleled recovery values and all data were corrected by the corresponding daily recovery factor for the feed type. Agreement between laboratories for mashes and pellets containing 0.0064, 0.0080, and 0.0096% lasalocid sodium was satisfactory. Coefficients of variation for mashes were 14.6, 9.8, and 10.7% and for pellets, 15,2, 10.9, and 15.6%. The average of the values found for the 3 mashes was 99.4% of the calculated value and for the 3 pellets, 86.5%. The method has been adopted as official first action.

Methods for the analysis of polyether antibiotics in animal tissues and fluids are described. For monensin and nigericin, only methods based on bioautography are available. For lasalocid, in addition to TLC bioautography for quantitation in chicken skin and fat, LC methods based on fluorescence detection have been developed for quantitation in animal blood, milk, liver, skin, and fat. In addition, a confirmatory method for lasalocid is described; this is based on purification by LC followed by silylation and pyrolysis gas chromatography-positive chemical ionization mass spectrometry.

A high pressure liquid chromatographic (HPLC) method has been developed to determine lasalocid in premixes. The method is simple and rapid, requiring an extraction of the drug and separation of insoluble material before HPLC. Elution times are typically less than 10 min per sample. The average recoveries for lasalocid at the 16.5 and 50% levels were 100.2 +/- 2 and 100.0 +/- 2%, respectively. The precision of the method (coefficient of variation = 0.02) compares favorably with that of the approved bioassay (coefficient of variation = 0.06).

A liquid chromatographic (LC) method with fluorometric detection was developed to quantitatively determine residue levels of monensin, salinomycin, narasin, and lasalocid in beef liver tissue. The ionophores are extracted from the tissue, purified by both alumina and Sephadex LH-20 column chromatography, and then derivatized. Lasalocid was directly esterified with 9-anthryldiazomethane (ADAM), but monensin, salinomycin, and narasin were first acetylated with acetic anhydride and then esterified with ADAM. The ADAM derivatives were purified on a silica gel column and separated by LC using an RP C-8 5 micron column. A fluorescence detector set at 365 nm (excitation) and 418 nm (emission) was used to monitor the column effluent. The detection limits were 0.15 ppm, and the calibration curves were linear between 0.5 and 5.0 ppm for all 4 ionophores. Mean recoveries were 57, 70, 75, and 90% for lasalocid (5 ppm), monensin (2.5 ppm), salinomycin (2.5 ppm), and narasin (2.5 ppm), respectively. The ionophores were also separated and semiquantitated by using bioautography and thin layer chromatography with a vanillin spray.

Lasalocid sodium is extracted directly from feeds and premixes with the mobile phase solution, an aliquot is chromatographed, and the concentration (peak height) of lasalocid sodium is measured by high pressure liquid chromatography with fluorescence detection. Chromatography and detection are completed in about 7 min. The results agree well with those found by the official AOAC fluorometric method, 42.A08--42.A13. The method offers an alternative to the AOAC method and has the capability of processing almost twice as many samples. The average recovery of 4 levels (0.0008, 0.0040, 0.0080, and 0.0120%) of lasalocid sodium from feeds is 100%. Replicate assays of typical poultry mashes and pellets containing about 0.0080% lasalocid sodium yielded coefficients of variation of 2.68--3.35%.

A collaborative study has been completed on a spectrofluorometric method for determining lasalocid sodium in finished poultry feeds. After a brief treatment of feed samples with pH 4.7 buffer, the drug is extracted with ethyl acetate. The ethyl acetate extract is further cleaned up by treatment with HCI and NaOH and quantitatively measured by spectrofluorometry. Nonspecific fluorescence is corrected for via the lasalocid-boric acid complex. The method is rapid, sensitive, and free of interference. A number of other feed additives, including monensin and ethoxyquin, do not interfere. Nine collaborators participated in the study and agreement between laboratories was satisfactory. The average recoveries of lasalocid sodium from the mash feeds added at levels of 0.0064, 0.0080, and 0.0096% were 100, 98, and 99%, respectively, and the corresponding coefficients of variation were 12.8, 8.2, and 8.0% (all results included). The method has been adopted as official first action.

The U.S. Food and Drug Administration (FDA) sponsored an interlaboratory study of a liquid chromatographic determinative procedure for lasalocid sodium in chicken skin with adhering fat. Four laboratories analyzed 35 dosed tissue samples and 82 fortified tissue samples containing lasalocid at levels ranging from 0.1 to 0.6 ppm. Samples were homogenized with acetonitrile, washed with hexane, and partitioned into the mobile phase prior to analysis liquid chromatography with fluorescence detection. The results of the interlaboratory study showed good reproducibility for fortified samples. Fortification levels, average recoveries, and interlaboratory percent coefficients of variation were as follows: 0.6 ppm, 0.57 ppm, and 9.7; 0.3 ppm, 0.25 ppm, and 9.1; and 0.15 ppm, 0.14 ppm, and 7.0, respectively. Data for analysis of the dosed tissue also showed good agreement among the laboratories.