Structural studies on the degradation products of vincristine dihydrogen sulfate

Vincristine was incubated at 37 degrees C for 72 h in 0.2 M glycine buffer (pH 7.4 or 8.8) containing 1% bovine serum albumin. The reaction mixture was extracted with CH2Cl2. High-performance liquid chromatography analysis (muBondapak C18 reverse-phase 10-micron steel column; solvent, 50% MeOH in 10 mM KH2PO4, pH 4.5; flow rate, 1.2 ml/min) of the CH2Cl2 extract gave 6 peaks, A, B, C, D, E, and F, with retention times of 4.8, 6.5, 10.0, 12.5, 17.5, and 23.5 min, respectively. Peak C corresponded with vincristine. Spectroscopic data for these peak fractions were as follows [UV (lambda max); infrared (cm-1); mass spectrum (m/z)]: peak A: 220, 256, and 295 nm; 3457, 2922, 1730, and 1669; and 783 (MH+); peak B: 218, 255, and 296 nm; 3435, 2922, 1731, and 1673; and 783 (MH+); peak C: 220, 255, and 296 nm; 3457, 2922, 1738, and 1680; and 825 (MH+); peak D: 218, 252, and 296 nm; 3385, 2922, 1734, and 1677; and 825 (MH+); peak E: 208, 218, 252, and 298 nm; 3371, 2922, 1727, and 1665; and 768 (MH2+); and peak F: 209, 222, 255, and 296 nm; 3392, 2922, 1734, and 1673; and 823 (MH+). These data suggest the following tentative structures for the degradation products: peak A, 4-deacetylvincristine; peak B, an isomer of 4-deacetylvincristine; peak D, an isomer of vincristine; peak E, 4-deacetyl-3-deoxyvincristine; and peak F, N-formylleurosine. The structure of peak A as 4-deacetylvincristine was confirmed by chemical synthesis.