GC-MS analysis of trimethylsilyl derivatives of amino-glycoside antibiotics

Kanamycin A, Kanamycin B and Neomycin B

Abstract

Trimethylsilyl derivatives (TMS) of Kanamycin A, Kanamycin B and Neomycin B were analysed by a GC-MS combined system. As the trimethyl silylation reagent, bis (trifluorosilyl) acetoamide (BSTFA) was used instead of tri-sil Z and N-trimethyl-silyldiethyl-amine.
Glass column 1m×3mmφ was packed with 1% OV-1 on silanized chromosorb W 80100 mesh. Because of the high operating temperature and to eliminate background peaks on mass spectra, the OV-1 column was conditioned approximately 5 days. Chromatogram of samples run at 250°C to 290°C (2°C/ min) of programed temperature. As the molecular ion of fully silylated samples were beyond the optimum capability of the LKB 9000 at 3.5kV of accelerating voltage, we used the 1.75 kV accelerating voltage. The mass spectra of the derivatives of Kanamycin A, Kanamycin B and Neomycin B were exhibited as minute molecular ion peaks at m/e 1276, m/e 1275 and m/e 1550. This means that all active hydrogens on both hydroxy and amine groups in Kanamycin A, and Kanamycin B and Neomycin B were completely silylated.
Fig. 1 shows the chromatogram of TMS samples. Fig. 2, 3 and 7 show the mass spectra of them and Fig. 5, 6 and 9 show the fragmentation schemes. The mass spectra of Kanamycin A exhibited strong intensities at m/e 810, m/e 723, m/e 450, m/e 360 and m/e 342 and Kanamycin B at m/e 810, m/e 809, m/e 722, m/e 450, m/e 449, m/e 360 and m/e 342. The difference of mass fragments of Kanamycin A and B appeared at the m/e 810, m/e 809, m/e 723, m/e 722, m/e 450 and m/e 449. This means that the difference of the -OTMS and the -NHTMS in Kanosamine ring is just one mass unit. The mass spectrum of Neomycin B exhibited strong intensities at m/e 1084, m/e 1010, m/e 741, m/e 725, m/e 449, m/e 377 and m/e 344. The absence of m/e 810 fragment ion (A-O-B.) is very interesting.
This method is a reliable technique for identifing the impurities and metabolites of antibiotics.