Development of a gas chromatography-mass spectrometry method for the analysis of aminoglycoside antibiotics using experimental design for the optimisation of the derivatisation reactions.

A packed column GC-electron-capture detection method for the analysis of the aminoglycoside antibiotics kanamycin and gentamicin was adapted to capillary GC-MS. The analytes were derivatised using a two-step procedure involving trimethylsilylation of the hydroxyl groups with trimethylsilylimidazole and acylation of the amino groups with heptafluorobutyrylimidazole. Electron impact mass spectra of the resulting derivatives of kanamycin A and gentamicins C1, C1a and C2 are given and interpreted. The derivatisation procedure was optimised using experimental design. This chemometrical approach considers main effects as well as interactions of the influential parameters, thus conducting a more thorough investigation of the method than the common step-by-step approach. Optimisation using fractional factorial and Box Behnken Designs produced a derivatisation method featuring better yield than previously published methods while in many cases requiring less reagents and shorter reaction times.

Isotope dilution GC-MS of benzylpenicillin residues in bovine muscle.

The German provisional prevalidated official method for the determination of residues of benzylpenicillin in muscle (beef) which uses gas chromatography with nitrogen-specific detection (GC-NPD) was successfully adapted to gas chromatography-mass spectrometry (GC-MS) with selected-ion monitoring. The programmed temperature vaporisation injection technique used in the original method was substituted by on-column injection. An isotope dilution procedure for benzylpenicillin using 13C2-labelled benzylpenicillin as internal standard was developed to improve the reliability of the GC-MS results. The spectral overlap of this labelled standard and the analyte caused a non-linear relationship in calibration for which linear bracketing and single-point calibration were compared. Single-point calibration proved to be the superior method, showing less deviation from the 'true' value, while at the same time requiring fewer calibration measurements. A comparison of the results obtained by using (i) external standardisation only, (ii) a close analogue of the analyte (phenoxymethylpenicillin) and (iii) 13C2-labelled Pen G as internal standard showed, as was to be expected, that varying recoveries are best corrected by using the isotope-labelled standard.