3.3.2.3 HPLC/MS experiments of hemin-artemisinin compounds
interactions
HPLC/MS analysis was performed on LS/MS/MS API 300. The
Column was a 4.6 x 250 mm (5m), SB-C18 Zorbax (Hewlett & Packard,
USA); the open temperature was 30oC. The eluents were A: H2O
adjusted to pH 3.2 by CH3COOH and B: methanol. The following gradient was
applied: from 50 % A and 50 % B to 25 % A and 75 % B within 70 minutes. Flow
elution was 1mL min-1, 20 L of samples were injected.
MS spectra were registered in positive and sometimes
in negative ion mode. The positive MS spectra were performed on an LCQ
electrospray directly coupled to the HPLC.
HPLC/DAD/MS analysis was performed on Agilent 1100
series LC/MSD Trap, under the same work conditions. Except this appliance is
equipped with UV DAD detector and UV-Vis spectra were recorded in the range
200-450nm. Products were detected at 412 nm in order to follow the modification
of the porphyrin chromophore.
All work solutions were mixed under magnetic stirring for 5
min and prepared, daily before each experiment or analysis and protected from
light. 2 M of hydrochloric acid and sodium hydroxide served to adjust the pH of
all solutions. 0.1 M tris (hydroxymethyl)-methylamine was used as buffer for
all solutions. For all HPLC-MS analysis, 1mL of 2 mM DMSO solution of
artemisinin compounds was mixed with 1 mL of 1 mM DMSO hemin solution and
incubated at (37 \u177\u177À1) o C over 10h.
| 