HPLC-UV detection of oxaliplatin, glutathione and degradation products in membrane vesicle incubation buffer.
Injection of blank membrane vesicle incubation buffer (A) and authentic standards showed chromatographic separation of glutathione (B, peak 1, retention time 6.5 min), oxaliplatin (C, peak 2, retention time 12.5 min) and Pt(DACH)Cl2 (D, peak 3, retention time 10.5 min) with no interference from components of the blank membrane vesicle incubation buffer. Oxaliplatin was incubated (100 μM, pH 7.4, 37˚C) in membrane vesicle incubation buffer with (I-L) or without glutathione (2 mM) (E-H) before analysis of samples by HPLC-UV after 0 hours (E,I), 0.3 hours (F,J), 2 hours (G,K) or 7 hours (H,L) incubation time. After 0.3 hours incubation time (F,J), HPLC-UV chromatograms appeared more-or-less unchanged from the start of the incubation (E,I) and similar with (J) and without glutathione (F). With an increasing incubation time, the oxaliplatin peak areas progressively reduced, and new peaks appeared, corresponding to Pt(DACH)Cl2 (G,H: peak 3) in solutions containing no glutathione, and an unknown peak (K,L: peak 4) in solutions containing glutathione. Chromatograms are representative of those from two independent experiments.