IDENTIFICATION AND DETERMINATION OF PIRLIMYCIN RESIDUE IN BOVINE-MILKAND LIVER BY HIGH-PERFORMANCE LIQUID-CHROMATOGRAPHY THERMOSPRAY MASS-SPECTROMETRY

Determinative and confirmatory methods of analysis for pirlimycin (I) residue in bovine milk and liver have been developed based on HPLC-the rmospray (TSP) MS. Milk sample preparation consisted of precipitating the milk proteins with acidified acetonitrile followed by a solvent pa rtitioning with a mixture of n-butyl chloride and hexane, extraction o f I from the aqueous phase into methylene chloride (MC), and solid-pha se extraction clean-up. For liver, samples (2 g) were extracted with 0 .25% trifluoroacetic acid in acetonitrile. The aqueous component was r eleased from the organic solvent with n-butyl chloride. The aqueous so lution was reduced in volume by evaporation, basified with ammonium hy droxide, then extracted with MC. The MC was evaporated to dryness and the dried residue reconstituted in 2.0 ml of 0.1 M ammonium acetate fo r analysis. A chromatographically resolved stereoisomer of I with TSP- MS response characteristics identical to I was used as an internal sta ndard (I.S.) for quantitative analysis based on the ratio of peak area s of I to I.S. in the protonated molecular-ion chromatogram at m/z 411 .2. The method for milk was validated by the analysis of control milk samples spiked with I at concentrations from 0.05 to 0.8 mu g/ml. The overall recovery of pirlimycin across this concentration range was 95. 4% +/- 8.7%. The limit of quantitation (LOQ) and limit of confirmation (LOC) of the method were validated to be 0.05 mu g/ml and 0.10 mu g/m l, respectively. The method for liver was validated by the analysis of control liver samples spiked with I at concentrations ranging from 0. 025 to 1.0 mu g/g. The overall recovery of pirlimycin was 97.6% +/- 5. 1% in this concentration range. The validated limit of quantitation (L OQ) and limit of configuration (LOC) of the method were 0.025 mu g/g a nd 0.10 mu g/g, respectively. Four diagnostic ions for I were monitore d for confirmation: the pseudo-molecular ions (M + H)(+) at m/z 411.2 (Cl-35) and m/z 413.2 (Cl-37) and fragment ions at m/z 375.2 and 158.1 . Confirmatory criteria were defined for these assays.