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A reverse-phase liquid chromatography/mass spectrometry (LC/MS) method, incorporating gradient elution, is described for the characterization of residual erythromycin A and its metabolites in salmon tissue. The method uses ion-spray, a mild atmospheric pressure ionization technique which provides an abundant protonated molecule well suited for selected ion monitoring experiments. Tandem mass spectrometry (MS/MS) using collision-induced dissociation was used to provide structural information. The LC/MS method was tested for the analysis of salmon tissue spiked with erythromycin A at levels between 0.01 and 1 p.p.m. A simple extraction and clean-up procedure, slightly modified from that described by Takatsuki et al. (J. Assoc. Off. Anal. Chem. 70, 708 (1987)), was used in this work. Using selected ion and selected reaction monitoring techniques, the LC/MS and LC/MS/MS methods provided detection limits of < 10 and 50 ng g-1, respectively. Confirmatory full-scan LC/MS and LC/MS/MS spectra were obtained at the 0.5 and 1 microgram g-1 levels, respectively. Using a combination of these techniques, the presence of residual erythromycin A was confirmed in the tissue of fish administered medicated feed containing the antibiotic. In addition, several metabolites and degradation products of erythromycin A, including anhydro-erythromycin and N-demethyl-erythromycin, were detected and where possible confirmed by comparison with authentic compounds. Although this analytical method has been shown to afford the necessary sensitivity and precision, application of these techniques to high-throughput quantitative analyses will require development of an improved clean-up procedure and preferably also of a suitable surrogate internal standard.

Six substitutional variants of gramicidin-S, present in a commercial extract of Bacillus brevis, have been characterized using a combination of liquid chromatographic/mass spectrometric analysis of the crude extract, plus accurate mass measurements, tandem mass spectrometric investigations, and amino acid analyses, of isolated high-performance liquid chromatographic fractions. Two of these variants were shown previously (Nozaki and Muramatsu, J. Antibiotics 37, 689 (1984)) to involve replacement of one or both Val residues in gramicidin-S by aminobutyric acid, and these findings are confirmed here. One of the four unknown variants corresponds to substitution of one Val residue by Leu, while the other three all involve substitution of one of the Orn residues in gramicidin-S by Lys, or by citrulline (gamma-carbamoyl ornithine), or by gamma-formyl ornithine. The biological activities of the purified fractions were assessed with respect to their in vitro inhibition of protein phosphatases PP1 and PP2A. For both protein phosphatases inhibition levels fell in the micromolar range, about four orders of magnitude higher than IC50 values for other small cyclic peptides such as microcystins and nodularins.